HK1128727B - Chimaeric fusion protein with superior chaperone and folding activities - Google Patents

Description

Chimeric fusion proteins with superior chaperone and folding activity

The present invention relates to the cloning, expression and use of a chimeric fusion protein having superior chaperone and folding activity compared to the naturally occurring counterpart. The present invention relates to chimeric fusion proteins encoded by recombinant DNA molecules comprising a nucleotide sequence encoding a non-human chaperone polypeptide binding segment and a nucleotide sequence encoding an FK506 binding protein (FKBP) or an FK 506-binding-protein-like domain (FKBP-like domain). In particular, the present invention relates to chimeric fusion proteins encoded by recombinant DNA molecules comprising a nucleotide sequence encoding a polypeptide binding segment of a non-human chaperone protein and a nucleotide sequence encoding a human FKBP-type peptidyl-proline cis-trans isomerase (PPIase), methods of producing these chimeric fusion proteins and their use as folding aids (helper) in the production of other proteins and in immunisation laboratory animals and in methods of vaccine or drug production, as fusion modules in recombinant protein technology and as folding aids for performing immunoassays.

Background

Molecular chaperones play an important role in a wide range of biotechnological applications today (Mogk et al, 2002 Chembiolchem 3, 807-). There are many folding aids that have chaperone as well as enzymatic properties. For this reason they can be used in many practical applications in the field of protein folding.

Chaperones, known as classical "folding aids", are polypeptides that assist in the folding of other proteins and maintain their structural integrity. They have the ability to facilitate folding of polypeptides both in vivo and in vitro. In general, folding aids are subdivided into folding catalysts and chaperones. The folding catalyst accelerates the rate-limiting step in protein folding due to its catalytic function. Examples of catalysts are described further below. Chaperonins are known to bind to denatured, partially denatured or hydrophobic surfaces of polypeptides and thereby aid in protein renaturation or keep them in solution. Thus, unlike folding catalysts, chaperones only exert a binding function (Buchner, J., Faseeb J10(1996) 10-19). Chaperones are ubiquitous stress-induced proteins involved in protein maturation, folding, transport and degradation (Gething, m.j. and Sambrook, j., Nature 355(1992) 33-45). Although they are also present under normal growth conditions, they are induced in large amounts under stress conditions. This further supports this view: their physiological function is to cope with stressful conditions.

To date, several different chaperone families are known. All of these chaperones are characterized by their ability to bind unfolded or partially unfolded proteins and have physiological functions associated with the proper folding of the protein or with the removal of denatured or aggregated proteins.

Examples of well-characterized chaperones are members of the so-called heat shock protein family, which are named according to their relative molecular weights, e.g. hsp100, hsp90, hsp70 and hsp60, and the so-called shsps (small heat shock proteins), as described in Buchner, J., Fasceb J10(1996)10-19 and Beissinger, M. and Buchner, J., biol. chem.379(1998) 245-59.

Unlike chaperones, folding catalysts assist folding by accelerating specific rate-limiting steps, thereby reducing the concentration of folding intermediates that are prone to aggregation. One class of catalysts, protein disulfide isomerases (otherwise known as thiol-disulfide-oxido-reductases), catalyze the formation or rearrangement of disulfide bonds in secreted proteins. In gram-negative bacteria, the oxidative folding of secreted proteins in the periplasm is regulated by a cascade of protein disulfide isomerases called DsbA, DsbB, DsbC and DsbD (Bardwell, J.C., Mol Microbiol 14(1994)199-205 and Missiakas, D.et al, Embo J14 (1995) 3415-24).

Another important class of folding catalysts known as peptidyl-proline cis-trans-isomerases (PPIs) includes different members such as CypA, PpiD (Dartigalongue, C. and Raina, S., Embo J17 (1998)3968-80, FkpA (Danese, P.N., et al, Genes Dev 9(1995)387-98), initiation factors (crook, E. and Wickner, W., Proc Natl Acad Sci U S A84 (1987)5216-20 and Stoller, G., et al, Embo J14 (1995)4939-48) and SlyD (Hottenrott, S., et al, J Biol Chem 272(1997) 15697-701).

Due to sequence similarity and protein topology, prolyl isomerase is divided into three distinct families: cyclophilin, FK506 binding protein (FKBP) and miniprotein. Cyclophilins bind to and are inhibited by the immunosuppressant, cyclosporin a. The parvoprotein is a prolyl isomerase family, which is inhibited by neither cyclosporin a nor FK 506. FKBP binds to and is inhibited by FK506 and rapamycin (the acronym FKBP stands for "FK 506-binding protein"; FK506 is a macrolide used as immunosuppressant drug). The first X-ray structure that FKBP is determined at high resolution is that of human FKBP 12. It is a five-stranded antiparallel beta-sheet wrapped around a short alpha-helix with a right-hand twist. The five-chain β -sheet framework comprises residues 2 to 8, 21 to 30, 35 to 38 and 46 to 49, 71 to 76 and 97 to 106(van Duyne et al Science (1991)252, 839-842). Subsequent studies have shown that FKBP, as well as cyclophilins and small proteins, form a highly conserved family of enzymes found in a wide range of prokaryotic and eukaryotic organisms (for review see John e.kay, biochem.j. (1996)314, 361-385). For example, 10 prolyl isomerases (2 parvoproteins, 3 cyclophilins and 5 FKBPs) have been identified in e.

Generally, FKBPs are defined according to binding criteria, i.e., they recognize and bind FK506 with high affinity in the nanomolar range. However, the presence of FKBP-like domains, which do not readily share significant sequence similarity, results in mutations at some of the amino acid residues that mediate FK506 binding and shifts affinity to the micromolar range. For example, SlyD and priming factors (two cytoplasmic ppiases from the cytoplasm of e.coli) can be considered FKBP-like proteins. Both prolyl isomerases contain domains sharing significant sequence homology with FKBP12, but their binding affinity to FK506 is rather weak and in the micromolar range (Scholz et al, Biochemistry (2006)45, 20-33). However, both SlyD and the trigger are undoubtedly members of the FKBP family in terms of sequence similarity and protein topology (Welfing et al, J.biol.chem (1994)269(4) 2895-.

The FKBP domains and FKBP-like domains may form part of a larger molecule with a complex topology. In mammalian cells, FKBP12, FKBP 12A and FKBP 13 comprise only the essential FKBP domain, whereas FKBP25 and FKBP52 have one or more FKBP domains as part of a larger molecule (for review see John e.kay, biochem.j. (1996)314, 361-385).

The modularly constructed FKBP has also been found in prokaryotic cells: for example, the aforementioned elicitor consists of three well-separated domains with distinct functions. The N-domain mediates binding to the E.coli ribosomal 50S subunit (Hesterkamp et al, J Biol Chem. (1997) Aug 29; 272 (35): 21865-71). The M (mid-) domain contains a prolyl isomerase active site (Stoller et al, FEBS Lett.1996 Apr 15; 384 (2): 117-22), and the C-domain includes a polypeptide binding site that mediates binding of an extended (extended) polypeptide substrate (Merz et al, J Biol chem.2006Oct 20; 281(42) 31963-31971). Another example of a modular constructed peptidyl-prolyl isomerase is periplasmic FkpA, which consists of an N-terminal chaperone and dimerization domain and a C-terminal FKBP-domain (Saul et al, J.mol.biol (2004)335, 595-608).

Some folding aids comprise both a catalytically active domain and a chaperone (or polypeptide binding) domain. For example, prolyl isomerase-initiating factors (Scholz et al, 1997, EmboJ.16, 54-58; Zarnt et al, 1997, JMB 271, 827-. It can recently be shown that FkpA and SlyD are very suitable as fusion modules for recombinant protein production. Both chaperones increase the expression rate of their client proteins, support correct refolding and increase the solubility of aggregation-prone (aggregation-pro) proteins such as retroviral surface proteins (Scholz et al, 2005, JMB 345, 1229-1241 and WO 03/000877).

FkpA, SlyD and SlpA are bacterial chaperones belonging to the FK506 binding protein (FKBP) family. As mentioned above, FK506 is a macrolide used as an immunosuppressant drug. The cellular receptor for FK506 is still in the focus of research groups worldwide. At the beginning of the nineties of the 20 th century, the three-dimensional structure of human FKBP, i.e., FKBP12, could be resolved (van Duyne et al, 1991, Science 252, 839-. In contrast to FkpA, SlyD and SlpA, human FKPB12 does not have any chaperone activity and it has only moderate prolyl isomerase activity.

Recombinantly produced proteins, such as antigens, are used in many diagnostic applications. These antigens may be produced as fusion proteins comprising a portion that constitutes an antigenic portion or target polypeptide that must be recognized by a specific binding partner present in the sample or assay mixture. Other portions of the recombinantly produced fusion protein are portions of the polypeptide that are fused to antigenic portions to facilitate cloning, expression, folding, solubilization, or purification of the particular antigen. The synthesis of recombinantly produced fusion proteins is well described in the prior art. It is common to use chaperones as part of a fusion protein that functions as accessory molecules for expression, folding, purification and solubilization of a target polypeptide. For example, U.S. Pat. No.6,207,420 discloses a fusion protein expression system for expressing heterologous proteins, i.e., amino acid sequences of a target polypeptide portion and a fusion peptide portion, which originate from different organisms. WO 03/000878 describes the use of FKBP chaperones as expression tools for retroviral surface glycoproteins.

Although the methods commonly used for expression, purification, folding and solubilization of fusion proteins appear to be operationally reliable, particularly those in which folding aids are used, there are still some problems that need to be solved. For example, whenever fusion proteins containing non-human amino acid sequences are used as binding partners in human diagnostic tests, interference problems can arise due to the use of these non-human proteins. It is often the case that a large number of antibodies present in a human blood sample react with bacterial proteins present in the assay reagents. Such interference may lead to high background noise or may even lead to erroneous test results. Another common problem is to change or optimize the affinity of the fusion partner to the respective client protein. The affinity of any fusion module for the target moiety must be well balanced. If the affinity is too high, the fusion protein will dissolve very well, but the complex between the fusion module and the client protein will maintain a closed conformation and will thus be inactivated in the immunoassay. If the affinity is too low, the client protein will be readily accessible and active in the immunoassay, but it will not be sufficiently protected from aggregation.

It is therefore an object of the present invention to provide an expression system suitable for the production of chaperone-like proteins which can be used in a wide range of biotechnological and especially diagnostic and pharmaceutical applications, which do not or only minimally interfere with molecules and substances present in isolated human samples. The prior art does not disclose an effective folding helper consisting mainly of human amino acid sequences, i.e. a helper that exerts both high catalytic and chaperone activities.

Despite the existence of several protein sequence alignments of human and bacterial chaperones (Walfing et al, 1994, JBC 269, 2895-.

Summary of The Invention

Surprisingly, we have been able to show that by fusing the polypeptide binding segment of a non-human chaperone protein to a sequence derived from an FK506 binding protein (FKBP) or an FKBP-like domain (FK 506-binding-protein-like domain), molecules with superior folding helper activity can be generated.

Specifically, by fusing the polypeptide binding segment of non-human chaperone proteins to sequences derived from FKBP-type human peptidyl proline cis-trans isomerase (PPIase), we were able to generate humanized PPIase chaperone molecules with folding helper activity superior to the wild-type folding helper. These chimeric humanized folding aids represent an extremely promising tool for the generation of native-like folded protein agents for a wide range of biotechnological applications, since they cause no or only little interference when used in diagnostic tests and pharmaceutical applications, and can be adapted to the respective protein due to their chaperone properties.

Also disclosed are preferred ways of designing recombinant DNA molecules encoding such chimeric fusion proteins and their use as part of an expression vector, host cells containing such expression vector and use in the production of chimeric fusion proteins.

Recombinantly produced chimeric fusion proteins which exhibit surprising and superior properties, especially with respect to their catalytic efficiency, are themselves part of the invention.

In further embodiments, the use of the recombinantly produced fusion protein as a folding aid for a target protein, as a folding aid in a process for producing a target protein, as an additive in an immunoassay cocktail, in a vaccine production process, for immunizing a laboratory animal and in a method for producing a medicament is disclosed.

Also disclosed are compositions comprising the recombinantly produced chimeric fusion proteins and a pharmaceutically acceptable excipient.

Brief Description of Drawings

FIG. 1: purification of FKBP12-IF1 (with SlyD insert) as demonstrated by SDS-PAGE. Lane 1, protein standard Mark 12 unstained from Invitrogen; lane 3, chaotropic crude lysate of overproduced E.coli strain BL21/DE 3; lane 5, IMAC flow through (flowthrough); lanes 7 to 11, imidazole elution fraction. FKBP12-IF1 can be purified and refolded in high yield in a simple one-step procedure as described in the examples section.

FIG. 2: near ultraviolet circular dichroism spectra of wild-type hFKBP12 (grey line) and hFKBP12-IF1 (black line) according to the invention. The buffer was 50mM sodium phosphate (pH7.5), 100mM NaCl, 1mM EDTA, and the protein concentration was 100. mu.M. The CD signal between 250 and 310nm reports the asymmetric environment of aromatic amino acid residues. The mean residue weight ovality (ovality) of hFKBP12 decreased after insertion into the SlyD IF loop. However, the reduced ovality was still directed to the compact native-like conformation of the FKBP12-IF1 chimera (black line).

FIG. 3: SlyD (1-165, SlyD) with and without an insert in the flap domain^*) Near ultraviolet circular dichroism spectrum. The buffer solution is 50mM sodium phosphate (pH7.5), 100mM NaCl, 1mM EDTA, and the protein concentration is 200. mu.M SlyD^*And 250. mu.M SlyD^*(Δ IF ring). SlyD^*Results in an average residue weight ovality of-40 deg cm at 278nm²dmol^-1(gray line). When the insert in the flap domain is removed, the shape of the near-UV CD signal is substantially preserved, but its intensity is increased (black line). This highlights that SlyD, after deletion of the IF loop domain^*The structural integrity of the structure is greatly maintained. In other words, it emphasizes the domain properties of the IF loop.

FIG. 4: in the presence of increasing concentrations of E.coli SlyD at 15 deg.C^*(1-165) refolding kinetics of RCM-T1. (A) Changes in fluorescence at 320nM, E.coli SlyD at 0, 3, 5, 8, 10, 15 and 20nM are shown^*Refolding kinetics in the presence of 100nM RCM-T1. (B) Slow folding Rate Pair SlyD^*Dependence on concentration. In SlyD^*Observed rate constant k in the Presence and absence_appAnd k₀Is shown as SlyD^*As a function of concentration. K is obtained from the slope of the line in (B)_cat/K_MValue of (2) 0.68X 10⁶M^-1s^-1. Refolding of RCM-T1 in 0.1M Tris-HCl (pH8.0) was initiated by dilution to 2.0M NaCl in the same buffer.

FIG. 5: refolding kinetics of RCM-T1 in the presence of increasing concentrations of the SlyD deletion variant SlyD (. DELTA.IF loop) at 15 ℃. (A) The refolding kinetics are shown for 100nM RCM-T1 in the presence of 0, 1.0, 2.0 and 5.0. mu.M SlyD (. DELTA.IF loop) from the change in fluorescence at 320 nM. (B) Dependence of the slow folding rate on the concentration of SlyD (Δ IF loop). Observed Rate constant k in the Presence and absence of SlyD (Δ IF Ring)_appAnd k₀The ratio of (d) is shown as a function of SlyD (Δ IF ring) concentration. The value of-500M was obtained from the slope of the line in (B)^-1s^-1. Refolding of RCM-T1 in 0.1M Tris-HCl (pH8.0) was initiated by dilution to 2.0M NaCl in the same buffer.

FIG. 6: the refolding kinetics of RCM-T1 in the presence of increasing concentrations of the human prolyl isomerase FKBP12 at 15 ℃. (A) The refolding kinetics are shown for 100nM RCM-T1 in the presence of 0, 0.5, 0.8, 1.0, 1.5 and 2.0. mu.M hFKBP12, based on the change in fluorescence at 320 nM. (B) Dependence of slow folding rate on hFKBP12 concentration. Observed rate constant k in the presence and absence of hFKBP12_appAnd k₀The ratio of (d) is shown as a function of hFKBP12 concentration. K is obtained from the slope of the line in (B)_cat/K_MValue of (2) 0.014X 10⁶M^-1s^-1. Tong (Chinese character of 'tong')Refolding of RCM-T1 in 0.1M Tris-HCl (pH8.0) was initiated by dilution to 2.0M NaCl in the same buffer.

FIG. 7: the refolding kinetics of RCM-T1 in the presence of increasing concentrations of the chimeric protein hFKBP12-IF1 according to the invention at 15 ℃. (A) The refolding kinetics for 100nM RCM-T1 in the presence of 0, 3, 5, 8, 10 and 20nM hFKBP12-IF1 are shown as a function of fluorescence at 320 nM. (B) Dependence of slow folding rate on concentration of hFKBP12-IF 1. Observed Rate constant k in the Presence and absence of hFKBP12-IF1_appAnd k₀The ratio of (A) is shown as a function of the concentration of hFKBP12-IF 1. K is obtained from the slope of the line in (B)_cat/K_MValue of (2.5X 10)⁶M^-1s^-1. Refolding of RCM-T1 in 0.1M Tris-HCl (pH8.0) was initiated by dilution to 2.0M NaCl in the same buffer.

FIG. 8: fusion protein SlyD^*-SlyD^*Schematic representation of-gp 41 and hFKBP12-IF1-hFKBP12-IF1-gp 41. Both the fusion module and gp41 are highlighted in boxes. Chaperone module SlyD^*And hFKBP12-IF1 were attached to the respective target molecules via a 23 amino acid flexible linker rich in glycine and serine residues. The hexa-histidine tag is fused to the C-terminus of the target molecule by a spacer segment, which improves accessibility and facilitates purification and refolding. The linker consists of five repeated GGGS elements (G: glycine, S: serine), and the spacer contains four HD repeats (H: histidine, D: aspartic acid) naturally occurring in the SlyD unstructured C-terminal tail.

FIG. 9: UV spectra of the chimeric fusion protein hFKBP12-IF1-gp41 according to the invention. Following matrix-coupled refolding and imidazole elution, the protein is soluble in aqueous buffer. To maintain absorption in the Lambert-Beer linear range, the protein stock was diluted 20-fold at room temperature to 5. mu.M in 50mM sodium phosphate (pH7.5), 100mM NaCl, 1.5mM EDTA. Protein aggregates and polymer conjugates are light stray particles that can lead to a skewed baseline in the wavelength region between 310 and 350 nm. The spectral shape demonstrates the absence of any aggregates and highlights the solubility of hFKBP12-IF1-gp 41.

FIG. 10: the refolding kinetics of RCM-T1 in the presence of increasing concentrations of the chimeric protein hFKBP12-IF4 (with SlpA insert) according to the invention at 15 ℃. (A) The refolding kinetics are shown for 100nM RCM-T1 in the presence of 0, 3, 6, 10, 15 and 20nM hFKBP12-IF4, as a function of fluorescence at 320 nM. (B) Dependence of slow folding rate on concentration of hFKBP12-IF 4. Observed Rate constant k in the Presence and absence of hFKBP12-IF4_appAnd k₀The ratio of (A) is shown as a function of the concentration of hFKBP12-IF 4. K is obtained from the slope of the line in (B)_cat/K_mValue of 850000M^-1s^-1. Refolding of RCM-T1 in 0.1M Tris-HCl (pH8.0) was initiated by dilution to 2.0M NaCl in the same buffer.

FIG. 11: the refolding kinetics of RCM-T1 in the presence of increasing concentrations of the chimeric protein hFKBP12-IF5 (carrying the Thermococcus FKBP18 insert) according to the invention at 15 ℃. (A) The refolding kinetics are shown for 100nM RCM-T1 in the presence of 0, 10, 25, 30, 35 and 40nM hFKBP12-IF5, as a function of fluorescence at 320 nM. (B) Dependence of slow folding rate on concentration of hFKBP12-IF 5. Observed Rate constant k in the Presence and absence of hFKBP12-IF5_appAnd k₀The ratio of (A) is shown as a function of the concentration of hFKBP12-IF 5. K is obtained from the slope of the line in (B)_cat/K_MValue of 660000M^-1s^-1. Refolding of RCM-T1 in 0.1M Tris-HCl (pH8.0) was initiated by dilution to 2.0M NaCl in the same buffer.

Brief description of the sequence listing

The attached sequence listing includes the following SEQ ID NOs:

SEQ ID NO.1 represents the amino acid sequence of E.coli SlyD according to Suzuki et al, 2003, JMB 328, 1149-1160, which is also available from the SwissProt database via IDP0A9K 9.

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDV

AVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVD

GNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGCCGG HGHDHGHEHG

GEGCCGGKGN GGCGCH

SEQ ID NO.2 shows the amino acid sequence of human FKBP12 (Suzuki et al, supra), which is also available from the SwissProt database via ID P62942.

GVQVETISPG DGRTFPKRGQ TCVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE

EGVAQMSVGQ RAKLTISPDY AYGATGHPGI IPPHATLVFD VELLKLE

SEQ ID NO.3 shows the amino acid sequence of human FKBP12 shown in SEQ ID NO.2 with a mutation at position 22. To achieve better solubility, cysteine 22 has been changed to alanine (C22A). In addition, a C-terminal hexahistidine tag has been added.

GVQVETISPG DGRTFPKRGQ TAVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE

EGVAQMSVGQ RAKLTISPDY AYGATGHPGI IPPHATLVFD VELLKLEHHH HHH

SEQ ID NO.4 shows the amino acid sequence of a preferred chimeric folding helper protein FKBP12-IF1 according to the invention. The SlyD insert is underlined.

FKBP12G1-G83/SlyD Q70-N129/FKBP12 L97-E107

GVQVETISPG DGRTFPKRGQ TCVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE

EGVAQMSVGQ RAKLTISPDY AYGQYDENLV QRVPKDVFMG VDELQVGMRF LAETDQGPVP

VEITAVEDDH VVVDGNHMLA GQNLVFDVEL LKLE

SEQ ID NO.5 shows the amino acid sequence of a preferred chimeric folding helper protein FKBP12-IF1 according to the invention. The SlyD insert is underlined. The sequence corresponds to SEQ ID NO.4, but cysteine 22 has been replaced by alanine.

FKBP12 G1-G83/slyD Q70-N129/FKBP12 L97-E107

GVQVETISPG DGRTFPKRGQ TAVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE

EGVAQMSVGQ RAKLTISPDY AYGQYDENLV QRVPKDVFMG VDELQVGMRF LAETDQGPVP

VEITAVEDDH VVVDGNHMLA GQNLVFDVEL LKLE

SEQ ID NO.6 shows the fusion protein SlyD^*-SlyD^*-amino acid sequence of gp41, which takes HIV-1gp41 polypeptide as target polypeptide and two SlyD^*The units fuse (compared to the state of the art). A schematic diagram of a vector-target type fusion protein is shown in fig. 8; see also example 1.

EcSlyD-[GGGS]₅GGG-EcSlyD-[GGGS]₅GGG-gp41(536-681；L555E，L566E，I573T，I580E)-HGHDHDHD-His6，pET24a

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE

GHEVGDKFDV AVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET

DQGPVPVEIT AVEDDHVVVD GNHMLAGQNL KFNVEVVAIR EATEEELAHG

HVHGAHDHHH DHDHDGGGSG GGSGGGSGGG SGGGSGGGKV AKDLVVSLAY

QVRTEDGVLV DESPVSAPLD YLHGHGSLIS GLETALEGHE VGDKFDVAVG

ANDAYGQYDE NLVQRVPKDV FMGVDELQVG MRFLAETDQG PVPVEITAVE

DDHVVVDGNH MLAGQNLKFN VEVVAIREAT EEELAHGHVH GAHDHHHDHD

HDGGGSGGGS GGGSGGGSGG GSGGGTLTVQ ARQLLSGIVQ QQNNELRAIE

AQQHLEQLTV WGTKQLQARE LAVERYLKDQ QLLGIWGCSG KLICTTAVPW

NASWSNKSLE QIWNNMTWME WDREINNYTS LIHSLIEESQ NQQEKNEQEL

LELDKWASLW NWFNITNWLW YHGHDHDHDH HHHHH

SEQ ID NO.7 shows the amino acid sequence of the chimeric fusion protein hFKBP12-IF1-hFKBP12-IF1-gp41, fused to hFKBP12-IF1 according to the invention with HIV-1gp41 polypeptide as target polypeptide (tandem fusion protein). A schematic of this protein is shown in figure 8; see also example 1.

MGVQVETISP GDGRTFPKRG QTAVVHYTGM LEDGKKFDSS RDRNKPFKFM

LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGQYDENL VQRVPKDVFM

GVDELQVGMR FLAETDQGPV PVE ITAVEDD HVVVDGNHML AGQNLVFDVE

LLKLEGGGSG GGSGGGSGGG SGGGSGGGGV QVETISPGDG RTFPKRGQTA

VVHYTGMLED GKKFDSSRDR NKPFKFMLGK QEVIRGWEEG VAQMSVGQRA

KLTISPDYAY GQYDENLVQR VPKDVFMGVD ELQVGMRFLA ETDQGPVPVE

ITAVEDDHVV VDGNHMLAGQ NLVFDVELLK LEGGGSGGGS GGGSGGGSGG

GSGGGTLTVQ ARQLLSGIVQ QQNNELRAIE AQQHLEQLTV WGTKQLQARE

LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLE QIWNNMTWME

WDREINNYTS LIHSLIEESQ NQQEKNEQEL LELDKWASLW NWFNITNWLW

YLEHHHHHH

SEQ ID NO.8 shows SlyD^*(SlyD 1-165) AmmoniaAmino acid sequence-corresponding to SEQ ID NO.1, but truncated after the C-terminal residue No. D165 (aspartic acid). Furthermore, SEQ ID NO.8 carries a hexahistidine tag at its C-terminus.

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDV

AVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVD

GNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDHHHHH H

SEQ ID NO.9 shows SlyD as shown in SEQ ID NO.8 without IF-loop^*(1-165) amino acid sequence. This variant is also referred to as SlyD^*A Δ IF-ring. For refolding and purification, it carries a hexa-histidine tag at its C-terminus.

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDV

AVGANDAYGA TGHPGIIPPH ATLKFNVEVV AIREATEEEL AHGHVHGAHD HHHDHDHDHH HHHH

SEQ ID NO.10 shows the amino acid sequence of the synthetic gene encoding the protein FKBP12-IF1 with a C-terminal hexahistidine tag to facilitate purification. The N-terminal methionine is cleaved off post-translationally by the bacterial N-methionyl-aminopeptidase, whereby the mature polypeptide is in fact initiated from glycine 1. When cysteine 22 was substituted by alanine, the resulting amino acid sequence of FKBP12-IF1 corresponded to SEQ ID NO. 5.

MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFM

LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGQYDENL VQRVPKDVFM

GVDELQVGMR FLAETDQGPV PVEITAVEDD HVVVDGNHML AGQNLVFDVE

LLKLEHHHHH H

SEQ ID NO.11 shows the FKBP12-IF1(C22A) -gp41 fusion construct (see also example 1).

MGVQVETI SP GDGRTFPKRG QTAVVHYTGM LEDGKKFDSS RDRNKPFKFM

LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGQYDENL VQRVPKDVFM

GVDELQVGMR FLAETDQGPV PVEITAVEDD HVVVDGNHML AGQNLVFDVE

LLKLEGGGSG GGSGGGSGGG SGGGSGGGTL TVQARQLLSG IVQQQNNELR

AIEAQQHLEQ LTVWGTKQLQ ARELAVERYL KDQQLLGIWG CSGKLICTTA

VPWNASWSNK SLEQIWNNMT WMEWDREINN YTSLIHSLIE ESQNQQEKNE

QELLELDKWA SLWNWFNITN WLWYLEHHHH HH

SEQ ID NO.12 shows the amino acid sequence of E.coli SlpA according to Suzuki et al, 2003, JMB 328, 1149-1160, which is also available via IDP0AEM0 from the SwissProt database. The N-terminal Met residue present in the unprocessed protein (not shown in SEQ ID NO. 12) is removed post-translationally. Until now, SlpA has very little information. Apart from being a preliminary feature of prolyl isomerase with rather low activity towards peptide substrates, virtually nothing has been known to SlpA so far.

SESVQSNSAV LVHFTLKLDD GTTAESTRNN GKPALFRLGD ASLSEGLEQH LLGLKVGDKT

TFSLEPDAAF GVPSPDLIQY FSRREFMDAG EPEIGAIMLF TAMDGSEMPG VIREINGDSI

TVDFNHPLAG QTVHFDIEVL EIDPALEA

SEQ ID NO.13 shows the amino acid sequence of a further preferred chimeric folding helper protein FKBP12-IF4 according to the invention. The SlpA insert is underlined. In addition, a C-terminal hexa-histidine tag was added.

FKBP12 G1-G83/S1pA V72-T132/FKBP12 L97-E107

GVQVETISPG DGRTFPKRGQ TAVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE

EGVAQMSVGQ RAKLTISPDY AYGVPSPDLI QYFSRREFMD AGEPEIGAIM LFTAMDGSEM

PGVIREINGD SITVDFNHPL AGQTLVFDVE LLKLEHHHHH H

SEQ ID No.14 shows the Pyrococcus FKBP18 amino acid sequence, which is also available from ID O93778 of the SwissProt database.

MKVEAGDYVL FHYVGRFEDG EVFDTSYEEI ARENGILVEE REYGPMWVRI GVGEIIPGLD

EAIIGMEAGE KKTVTVPPEK AYGMPNPELV ISVPREEFTK AGLEPQEGLY VMTDSGIAKI

VSVGESEVSL DFNHPLAGKT LVFEVEVIEV KKAEEDSEA

SEQ ID NO.15 shows the amino acid sequence of a further preferred chimeric folding helper protein FKBP12-IF5 according to the invention. The Pyrococcus FKBP18 insert is underlined. In addition, a C-terminal hexa-histidine tag was added.

FKBP12 G1-G83/TcFKBP18 M84-T140/FKBP12 L97-E107

GVQVETISPG DGRTFPKRGQ TCVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE

EGVAQMSVGQ RAKLTISPDY AYGMPNPELV ISVPREEFTK AGLEPQEGLY VMTDSGIAKI

VSVGESEVSL DFNHPLAGKT LVFDVELLKL EHHHHHH

SEQ ID NO.16 shows the amino acid sequence of the E.coli initiation factor, which is also available from ID P0A850 of the SwissProt database.

MQVSVETTQG LGRRVTITIA ADSIETAVKS ELVNVAKKVR IDGFRKGKVP MNIVAQRYGA

SVRQDVLGDL MSRNFIDAII KEKINPAGAP TYVPGEYKLG EDFTYSVEFE VYPEVELQGL

EAIEVEKPIV EVTDADVDGM LDTLRKQQAT WKEKDGAVEA EDRVTIDFTG SVDGEEFEGG

KASDFVLAMG QGRMIPGFED GIKGHKAGEE FTIDVTFPEE YHAENLKGKA AKFAINLKKV

EERELPELTA EFIKRFGVED GSVEGLRAEV RKNMERELKS AIRNRVKSQA IEGLVKANDI

DVPAALIDSE IDVLRRQAAQ RFGGNEKQAL ELPRELFEEQ AKRRVVVGLL LGEVIRTNEL

KADEERVKGL IEEMASAYED PKEVIEFYSK NKELMDNMRN VALEEQAVEA VLAKAKVTEK

ETTFNELMNQ QA

SEQ ID NO.17 shows the FKBP domain of the E.coli initiation factor according to SEQ ID NO. 16. Amino acids methionine 140 to glutamic acid 251 belong to the FKBP domain of the initiating factor.

MLDTLRKQQA TWKEKDGAVE AEDRVTIDFT GSVDGEEFEG GKASDFVLAM GQGRMI PGFE

DGIKGHKAGE EFTIDVTFPE EYHAENLKGK AAKFAINLKK VEERELPELT AE

SEQ ID NO.18 shows an amino acid sequence of yet another embodiment of the present invention. In this chimeric folding helper protein (elicitor-IF/SlyD), the IF domain derived from SlyD is inserted into the FKBP domain of E.coli elicitor. The SlyD insert is underlined.

E.coli initiation factor/FKBP-+IF

TF M140-H222/SlyD Q70-N129/TF A231-E251

MLDTLRKQQA TWKEKDGAVE AEDRVTIDFT GSVDGEEFEG GKASDFVLAM GQGRMIPGFE

DGIKGHKAGE EFTIDVTFPE EYHQYDENLV QRVPKDVFMG VDELQVGMRF LAETDQGPVP

VEITAVEDDH VVVDGNHMLA GQNAKFAINL KKVEERELPE LTAE

SEQ ID NO.19 shows the amino acid sequence of the unprocessed precursor FkpA from E.coli. The newly translated FkpA carries the N-terminal signal sequence (Met 1-Ala25) for export to the periplasm. After passage through the inner membrane, the signal peptidase specifically removes the signal sequence, whereby this sequence is deleted in the processed functional protein. FkpA contains an N-terminal chaperone and dimerization domain as well as a C-terminal isomerase domain (Gly 147-K249). In the RNase T1 test, FkpA showed about 250,000M^-1s^-1The catalytic efficiency of (a). The FkpA sequence can also be determined by SwissProt ID: p45523.

MKSLFKVTLL ATTMAVALHA PITFAAEAAK PATAADSKAA FKNDDQKSAY ALGASLGRYM

ENSLKEQEKL GIKLDKDQLI AGVQDAFADK SKLSDQEIEQ TLQAFEARVK SSAQAKMEKD

AADNEAKGKE YREKFAKEKG VKTSSTGLVY QVVEAGKGEA PKDSDTVVVN YKGTLIDGKE

FDNSYTRGEP LSFRLDGVIP GWTEGLKNIK KGGKIKLVIP PELAYGKAGV PGIPPNSTLV

FDVELLDVKP APKADAKPEA DAKAADSAKK

SEQ ID NO.20 shows the amino acid sequence of the FKBP domain of FkpA as shown in SEQ ID NO.19 (G147-K249). The C-terminal sequence LE is included due to the cloning strategy. A C-terminal hexahistidine tag has been added to facilitate purification. It is hypothesized that this FKBP domain has a weak activity in the RNase T1 folding assay, which is intermediate to SlyD^*Between the Δ IF loop and human FKBP12 (see table 1).

GLVYQVVEAG KGEAPKDSDT VVVNYKGTLI DGKEFDNSYT RGEPLSFRLD GVIPGWTEGL

KNIKKGGKIK LVIPPELAYG KAGVPGIPPN STLVFDVELL DVKPAPLEHH HHHH

SEQ ID NO.21 shows an amino acid sequence according to yet another embodiment of the present invention. In this chimeric folding helper protein FKpA-IF/SlyD, the IF domain derived from SlyD is inserted into the FKBP domain of FkpA (as shown in SEQ ID NO. 20). A C-terminal hexahistidine tag has been added to facilitate purification. The chimeric folding helper protein is expected to show high activity in the rnase T1 folding assay.

FkpA G147-G226/SlyD Q70-N129/FkpA L239-P252

GLVYQVVEAG KGEAPKDSDT VVVNYKGTLI DGKEFDNSYT RGEPLSFRLD GVIPGWTEGL

KNIKKGGKIK LVIPPELAYG QYDENLVQRV PKDVFMGVDE LQVGMRFLAE TDQGPVPVEI

TAVEDDHVVV DGNHMLAGQN LVFDVELLDV KPAPLEHHHH HH

Detailed description of the invention

The present invention relates to recombinant DNA molecules encoding chimeric fusion proteins comprising:

a) at least one nucleotide sequence encoding a non-human chaperone polypeptide binding segment

b) At least one upstream nucleotide sequence coding for an FK 506-binding protein (FKBP) or an FK 506-binding-protein-like domain (FKBP-like domain) and

c) at least one nucleotide sequence encoding an FK 506-binding protein (FKBP) or an FK 506-binding-protein-like domain (FKBP-like domain) downstream thereof. The nucleotide sequence of a) on the one hand and the nucleotide sequences of b) and c) on the other hand may originate from the same organism, but they must encode different parental FKBP molecules. More specifically, a) encodes a chaperone domain of one FKBP molecule (e.g. SlyD or SlpA), and b) and c) encode an FKBP domain or FKBP-like domain of another molecule (e.g. human FKBP 12). The nucleotide sequences encoding the FK506 binding protein (FKBP) or the FK 506-binding-protein-like domain (FKBP-like domain), i.e. those of part b) and part c), may originate from the same organism, but they may also originate from different organisms. Preferably, the sequences given under b) and c) originate from the same organism. More preferably, they are derived from the same parent FKBP molecule, e.g., human FKBP 12.

In particular, the present invention relates to recombinant DNA molecules encoding chimeric fusion proteins comprising:

a) at least one nucleotide sequence encoding a non-human chaperone polypeptide binding segment

b) At least one nucleotide sequence coding for FKBP type human peptidyl proline cis-trans isomerase (PPIase) upstream thereof and

c) at least one nucleotide sequence for coding FKBP type human peptide base proline cis-trans isomerase (PPIase) at the downstream.

The term "recombinant DNA molecule" refers to a DNA molecule obtained by combining two otherwise separate segments of sequence by manual manipulation of the isolated polynucleotide segments, either by genetic engineering techniques or by chemical synthesis. In doing so, one can join together polynucleotide segments having the desired function to produce a combination of the desired functions.

The term "chimeric fusion protein" means that the polypeptide binding domain and the FKBP (or FKBP-like) domain are derived from different parental molecules. We view FKBP or FKBP-like domains as folding scaffolds onto which chaperone domains can be grafted to produce superior chaperones with superior folding helper activity. In the present invention, the non-human polypeptide is fused to a human polypeptide sequence. Chimeric proteins may also be referred to as "mosaic proteins". Since it is an object of the present invention to humanize the folding helper protein such that the resulting protein is more tolerable in diagnostic applications, the percentage of the non-human amino acid sequence is preferably no more than fifty percent of the portion compared to the length of the complete chimeric fusion protein.

Preferably, the nucleotide sequences according to a), b) and c) are not separated by an additional linker sequence, but are directly adjacent to one another.

By "upstream" direction is meant that the nucleotide is located 5' to the polynucleotide, i.e., toward the first nucleotide. The term "upstream" in terms of amino acid sequence means that the amino acid is located in the N-terminal direction, i.e.towards the beginning of the polypeptide.

By "downstream" direction is meant that the nucleotide is located 3' to the polynucleotide, i.e., toward the last nucleotide. The term "downstream" in terms of amino acid sequence means that the amino acids are located in the C-terminal direction, i.e.towards the end of the polypeptide.

A polynucleotide is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or fragment thereof in its native state or when manipulated by methods known in the art.

A "polypeptide binding segment" of a chaperone is considered to be the portion of the chaperone that binds and retains a polypeptide chain during three-dimensional folding of the protein. The "polypeptide binding segment" of the E.coli chaperone SlyD, i.e.its chaperone properties, has been mapped in this application to the so-called IF domain (insert flap domain, amino acids 76-122). As an autonomous folding unit, a protein domain is capable of adopting a native-like stable fold in aqueous solution. The terms "polypeptide binding segment", "IF-loop", IF-domain or chaperone domain may be used synonymously.

Preferred non-human chaperones are E.coli SlyD and SlpA and FKBP chaperones of archaea such as FKBP 17 from Methanococcus thermoautotrophic (Methanococcus thermoautotrophicus), FKBP18 from Methanococcus jannaschii (Methanococcus jannaschii), FKBP18 from Thermococcus species (Thermoccussp.) KS1, FKBP29 from Pyrococcus horikoshii, FKBP26 from Methanococcus jannaschii and FKBP30 from Thermomyces facilis (Aeropyrum pernix) as listed by Suzuki et al, 2003, JMB 328, 1149-1160.

In a preferred embodiment, the at least one nucleotide sequence encoding a non-human chaperone polypeptide binding segment comprises a sequence encoding a non-human FK506 binding protein (FKBP). More preferred are the FKBP sequences of E.coli, Methanococcus thermoautotrophic, Methanococcus jannaschii, Pyrococcus species KS1, Pyrococcus horikoshii or Thermus facilis, and E.coli SlyD and SlpA sequences are most preferred.

In a particularly preferred embodiment, the E.coli SlyD sequence comprises a nucleotide sequence encoding a polypeptide which starts at the N-terminus with any amino acid located between amino acids 56 and 75 of SEQ ID NO.1 and ends at the C-terminus with any amino acid located between amino acids 122 and 136 of SEQ ID NO. 1. Most preferred is a sequence encoding a polypeptide which starts at amino acid 70 of SEQ ID NO.1 at the N-terminus and ends at amino acid 129 of SEQ ID NO.1 at the C-terminus.

In a further preferred embodiment, the E.coli SlyA sequence comprises a nucleotide sequence encoding a polypeptide which starts at the N-terminus with any amino acid located between amino acids 56 to 75 of SEQ ID NO.12 and ends at the C-terminus with any amino acid located between amino acids 122 to 136 of SEQ ID NO. 12. Most preferred is a sequence encoding a polypeptide which starts at amino acid 72 of SEQ ID NO.12 at the N-terminus and ends at amino acid 132 of SEQ ID NO.12 at the C-terminus.

As for upstream and downstream sequences adjacent to the nucleotide sequence encoding the non-human chaperone polypeptide binding segment, these upstream and downstream sequences originate from the FK506 binding protein or FK 506-binding-protein-like domain (also referred to as FKBP-like domain).

According to the present invention, FK506 binding proteins (FKBPs) are proteins that are able to recognize and bind the immunosuppressant FK506 with high affinity in the nanomolar range. FKBP-like domains ("FK 506-binding-protein-like domains") or FKBP-like proteins are proteins or protein parts which are no longer susceptible or hardly susceptible to inhibition of prolyl isomerase by FK 506. These FKBP-like domains share considerable sequence and structural similarity with FK506 binding proteins like FKBP12, but some of the amino acid residues that mediate FK506 binding are mutated and the affinity shifted to the micromolar range. For example, SlyD and a priming factor, two PPIases from E.coli cytoplasm, were considered FKBP-like proteins (Callebout & Mornon, FEBS Lett. (1995)374(2) 211-; Hulfing et al, J.biol. chem. (1994)269(4), 2895- & 2901)).

In the case of the upstream and downstream sequences adjacent to the nucleotide sequence encoding a polypeptide binding segment of a non-human chaperone protein like e.coli SlyD or SlpA, it is preferred that the upstream and/or downstream nucleotide sequence encoding an FKBP-type human peptidyl proline cis-trans isomerase comprises a nucleotide sequence encoding an FK506 binding protein (FKBP) or an FKBP-like domain, with the sequence encoding human FKBP12 being particularly preferred.

Further in a preferred embodiment, the upstream nucleotide sequence encoding FKBP12 comprises a sequence encoding a polypeptide which starts at the N-terminus with any amino acid located between amino acids 1 to 20 of SEQ ID NO.3 and ends at the C-terminus with any amino acid located between amino acids 70 to 89 of SEQ ID NO. 3.

Also preferred is an embodiment wherein the downstream nucleotide sequence encoding FKBP12 comprises a sequence encoding a polypeptide which N-terminally starts at any one of the amino acids located between amino acids 90 to 97 of SEQ ID No.3 and C-terminally ends at any one of the amino acids located between amino acids 103 to 107 of SEQ ID No. 3.

Most preferred are recombinant DNA molecules comprising a nucleotide sequence encoding a polypeptide according to SEQ ID No. 4. SEQ ID NO.4 shows the amino acid sequence starting at the N-terminus at amino acid positions glycine/G1 to glycine/G83 (FKBP12) of SEQ ID NO.3, followed by the amino acid positions glutamine/Q70 to asparagine/N129 (SlyD) of SEQ ID NO.1 and ending with leucine/L97 to glutamic acid/E107 (FKBP12) of SEQ ID NO. 3. The polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.4 is also referred to as FKBP12-IF 1.

In a further preferred embodiment of the invention, the recombinant DNA molecule comprises a nucleotide sequence encoding a polypeptide according to SEQ ID No. 13. SEQ ID NO.13 shows the amino acid sequence starting at the N-terminus at amino acid positions glycine/G1 to glycine/G83 (FKBP12) of SEQ ID NO.3, followed by amino acid positions valine/V72 to threonine/T132 (SlyA) of SEQ ID NO.12 and ending with leucine/L97 to glutamic acid/E107 (FKBP12) of SEQ ID NO. 3. The polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.13 is also referred to as FKBP12-IF 4.

In a further preferred embodiment of the invention, the recombinant DNA molecule comprises a nucleotide sequence encoding a polypeptide according to SEQ ID No. 15.

SEQ ID NO.15 shows the amino acid sequence starting at the N-terminus at amino acid positions glycine/G1 to glycine/G83 (FKBP12) of SEQ ID NO.3, followed by amino acid positions methionine/M84 to threonine/T140 (Pyrococcus FKBP18) of SEQ ID NO.14 and ending with leucine/L97 to glutamic acid/E107 (FKBP12) of SEQ ID NO. 3. The polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.15 is also referred to as FKBP12-IF 5.

It is advantageous to select DNA sequences inserted upstream and downstream of the sequence encoding the non-human chaperone polypeptide binding segment in such a way that: two-dimensional structural elements such as beta-sheets are not interrupted by heterologous sequence elements and remain intact. Commonly known sequence alignments assist in the selection of appropriate upstream and downstream sequences, as for example, Suzuki et al, 2003, JMB 328, 1149-1160.

According to the present invention, the selection and arrangement of the nucleotide sequence encoding the non-human chaperone polypeptide binding segment and the upstream and downstream nucleotide sequences, i.e. the nucleotide sequence encoding the FK506 binding protein (FKBP) or the FK 506-binding-protein-like domain (FKBP-like domain), and preferably the nucleotide sequence encoding the FKBP-type human peptidyl proline cis-trans isomerase (PPIase), is done in such a way that: the overall structural order of the resulting chimeric fusion protein corresponds to the structure of a naturally occurring chaperone. In other words, the overall structure preferably maintains an arrangement of secondary structural elements like alpha-helices and beta-sheets as described in the state of the art (e.g., Suzuki et al, 2003, JMB 328, 1149-.

The invention specifically relates to chimeric fusion proteins produced by expression of such recombinant DNA molecules.

By expression of the recombinant DNA molecules identified above, we have been able to provide the counterparts of the bacterial PPIase chaperones SlyD, FkpA, the trigger factor and SlpA, and even the humanized counterparts of the bacterial PPIase chaperones SlyD, the trigger factor and SlpA. These humanized peptidyl proline cis-trans isomerase chaperones can serve as a beneficial tool in biotechnological applications and as an additive in diagnostic tests. As can be seen in the experimental part of the present application, we have been able to obtain humanized chaperones with higher catalytic efficiency than the wild-type folding helper whose amino acid sequence is comprised in the humanized chaperones of the present invention. Based on observations in the rnase T1 refolding test system showing the folding and refolding ability of proteins, we have been able to show that isolated human FKBP12 has only about 14,000M^-1s^-1Small catalytic efficiency. The catalytic efficiency of the separated unmodified escherichia coli SlyD reaches 680,000M^-1s^-1. Deletion variants of SlyD lacking the IF loop domain showed 500M in the RNase T1 folding assay^-1s^-1Negligible catalytic efficiency. Surprisingly, the catalytic efficiency of the chimeric molecule FKBP12-IF1 (amino acid sequence shown in SEQ ID NO.4) was much greater than this value. FKBP12-IF1 showed about 2,500,000M^-1s^-1(see also table 1 in the examples section) which exceeds the values of the most effective prolyl isomerases known to date. This value even exceeds the catalytic efficiency of the initiating factor, which is equal to 1.2X 10⁶M^-1s^-1(Stoller et al (1995) EMBO J.14, 4939-.

By combining the active site of the prolyl isomerase center of human FK506 binding proteins with the polypeptide binding domain of non-human chaperones (the so-called IF loop), we have generated folding helpers with superior chaperone and enzymatic properties. We have been able to provide folding aids with higher catalytic efficiency than the isolated wild-type protein. The folding aids according to the invention can therefore also be referred to as super chaperones (super chaperones) in respect of their excellent catalytic efficiency.

Thus part of the invention is a recombinantly produced fusion protein comprising a) a polypeptide sequence comprising a polypeptide binding segment of a non-human chaperone protein, b) a polypeptide sequence of an FK506 binding protein (FKBP) or an FK 506-binding-protein-like domain (FKBP-like domain) fused to the N-terminus of the non-human chaperone polypeptide sequence, and C) a polypeptide sequence of an FK506 binding protein (FKBP) or an FK 506-binding-protein-like domain (FKBP-like domain) fused to the C-terminus of the non-human chaperone polypeptide sequence.

Thus one preferred embodiment is a recombinantly produced fusion comprising: a) a polypeptide sequence comprising a polypeptide binding segment of a non-human chaperone protein, b) a human FKBP-type peptidyl proline cis-trans isomerase polypeptide sequence fused to the N-terminus of the non-human chaperone polypeptide sequence, C) a human FKBP-type peptidyl proline cis-trans isomerase polypeptide sequence fused to the C-terminus of the non-human chaperone polypeptide sequence. A preferred embodiment is a chimeric fusion protein comprising a polypeptide binding segment of the E.coli SlyD chaperone sequence and a human FKBP12 polypeptide sequence fused N-and C-terminally to the SlyD sequence. One of the preferred embodiments of the present invention is a chimeric fusion protein comprising an amino acid sequence according to SEQ ID No. 4.

Also preferred are chimeric fusion proteins comprising a polypeptide binding segment of the e.coli SlpA chaperone sequence and a human FKBP12 polypeptide sequence fused to the SlpA sequence at the N-and C-terminus. More details of chimeric fusion proteins comprising polypeptide binding segments of the e.coli SlpA chaperone sequence are given in the examples section and table 1.

A preferred embodiment of the present invention is a chimeric fusion protein comprising an amino acid sequence according to SEQ ID NO. 13. The protein is named FKPB12-IF 4.

Also preferred are fusion proteins comprising a chaperone sequence polypeptide binding segment of the Pyrococcus FKBP18 protein and a sequence of a human FKBP12 polypeptide fused N-and C-terminally to the Pyrococcus FKBP18 sequence. Thermococcus FKBP18 is a thermostable homolog of SlyD that carries an IF domain in the flap region near the active site of prolyl isomerase. The amino acid sequence of Pyrococcus FKBP18 is shown in SEQ ID NO. 14. The amino acid sequence of the resulting chimeric fusion protein is shown in SEQ ID NO.15, in which the putative IF loop domain of Thermococcus FKBP18 was grafted onto the folding scaffold of hFKBP 12. More details about the chimeric fusion protein are given in the examples section and table 1.

In another embodiment of the invention, an IF domain derived from SlyD is inserted into the FKBP domain of E.coli initiation factor. SEQ ID NO.18 shows the amino acid sequence of the chimeric folding helper protein (elicitor-IF/SlyD). The IF domain derived from SlyD was inserted into the FKBP domain of E.coli initiation factor. The SlyD insert is underlined.

In yet another embodiment, an IF domain derived from SlyD is inserted into the FKBP domain of FkpA (shown in SEQ ID NO. 20). The resulting chimeric folding helper protein is called FkpA-IF/SlyD. The chimeric folding helper protein is expected to show high activity in the rnase T1 folding assay. SEQ ID NO.21 shows the amino acid sequence of a chimeric folding helper protein according to the invention.

From our experiments we concluded that chaperone functions of SlyD, SlpA and TcFKBP18 are restricted to the so-called if (insert in flap) domain. We conclude that the IF domains of different FKBP-chaperones are structurally related and functionally equivalent. Therefore, IF domains from different FKBP-chaperones should be interchangeable. We hypothesized that the IF domain within SlyD can be replaced by a putative IF domain, for example in SlpA or TcFKBP18, without compromising the true folding helper properties of SlyD. This chaperone domain exchange should be possible with each other, i.e. the putative IF domain can be grafted onto the FKBP-like domain of SlpA or TcFKBP18 to generate a functional chaperone module. The interchange of chaperone domains can pave the way for custom folding aids with substrate affinities appropriate for the respective target proteins.

In FKBP-X fusion proteins, FKBP functions as a carrier module, and X means a guest or target protein, the carrier module and the guest protein being present in a dynamic equilibrium in closed and open form. In the closed form, the hydrophobic region is masked and thus the fusion protein remains soluble without aggregation. In the open form, antigenic sites are exposed, which makes the guest protein functional, for example, in immunoassays. The affinity must therefore be well balanced in the fusion protein and it can be adapted to the needs of the target module by interchanging the IF-domains from different FKBP-chaperones.

Optionally all chimeric fusion proteins may be further fused to the target polypeptide sequence. The target polypeptide according to the invention may be any polypeptide that is desired in large quantities and is therefore difficult to isolate or purify from other non-recombinant sources.

Examples of target proteins preferably produced by the methods of the invention include mammalian gene products such as enzymes, cytokines, growth factors, hormones, vaccines, antibodies, and the like. More specifically, preferred overexpressed gene products of the invention include, for example, erythropoietin, insulin, growth hormone releasing factor, platelet derived growth factor, epidermal growth factor, transforming growth factor alpha, transforming growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor I, insulin-like growth factor II, coagulation factor VIII, superoxide dismutase, interferon, y-interferon, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, granulocyte colony stimulating factor, multispectral colony stimulating factor, granulocyte-macrophage stimulating factor, macrophage colony stimulating factor, erythropoietin, human immunodeficiency virus, human immunodeficiency, Gene products such as T cell growth factor, lymphotoxin, and the like. Preferred overexpressed gene products are human gene products.

Furthermore, the present methods can be readily adapted to enhance secretion of any over-expressed gene product that can be used as a vaccine. Overexpressed gene products useful as vaccines include any structural, membrane-associated, or secreted gene product of a mammalian pathogen. Mammalian pathogens include viruses, bacteria, single-or multi-cell parasites that can affect or attack mammals. For example, viral vaccines can include vaccines against viruses such as Human Immunodeficiency Virus (HIV), vaccinia, poliovirus, adenovirus, influenza, hepatitis a, hepatitis B, dengue virus, japanese B encephalitis, varicella zoster, cytomegalovirus, hepatitis a, rotavirus, and viral diseases such as measles, yellow fever, mumps, rabies, herpes, influenza, parainfluenza, and the like. Bacterial vaccines may include vaccines against bacteria such as Vibrio cholerae (Vibrio cholerae), Salmonella typhi (Salmonella typhi), Bordetella pertussis (Bordetella pertussis), Streptococcus pneumoniae (Streptococcus pneumoniae), haemophilus influenzae (haemophilus influenzae), Clostridium tetani (Clostridium tetani), Corynebacterium diphtheriae (Corynebacterium diphtheriae), Mycobacterium leprae (Mycobacterium leprae), rickettsia rickettsii (r.rickettsii), Shigella (Shigella), gonococcus (Neisseria norrhae), Neisseria meningitidis (neisserial meningitidis), coccidioidea (coccidisis), borrelia burgdorferi (nei), and the like. Preferably, the target protein is a member of the group consisting of retroviral proteins such as gp41 and p17 from HIV-1, gp36 and p16 from HIV-2, gp21 from HTLV-I/II, viral envelope proteins such as E1 and E2 from rubella virus, or amyloidogenic proteins such as beta-AP 42 (Alzheimer's peptide) or prion proteins.

The target polypeptide according to the invention may also contain sequences from several different proteins, e.g. diagnostically relevant epitopes, which are constructed to be expressed as a single recombinant polypeptide.

Recombinant DNA molecules which code for chimeric fusion proteins and which contain a) at least one nucleotide sequence which codes for a polypeptide binding segment of a non-human chaperone protein, b) at least one nucleotide sequence which codes for an FK 506-binding protein or an FK 506-binding-protein-like domain (FKBP-like domain) upstream thereof and c) at least one nucleotide sequence which codes for an FK 506-binding-protein-like domain (FKBP-like domain) downstream thereof and d) at least one nucleotide sequence which codes for a target polypeptide are also an object of the present invention.

Preferably, a recombinant DNA molecule encoding a chimeric fusion protein comprising a) at least one nucleotide sequence encoding a polypeptide binding segment of a non-human chaperone protein, b) at least one nucleotide sequence encoding an FKBP-type human peptidyl proline cis-trans isomerase (PPIase) upstream thereof and, c) at least one nucleotide sequence encoding an FKBP-type human peptidyl proline cis-trans isomerase (PPIase) downstream thereof and d) at least one nucleotide sequence encoding a target polypeptide is also an object of the present invention.

It is important that the nucleotide sequence encoding the target polypeptide is inserted in such a way that: the sequence encoding the chimeric super chaperone according to steps a), b) and c) remains intact so that it maintains its catalytic and chaperone functions. This means that the nucleotide sequence encoding the target polypeptide is inserted in-frame upstream or downstream of the sequence encoding the chimeric fusion protein. It may be inserted upstream and downstream, and also more than one copy.

The recombinant DNA encoding the chimeric fusion protein and the target polypeptide according to the invention may also contain a linker sequence which results in the production of a linker polypeptide upon expression of the complete protein. As will be appreciated by those skilled in the art, such linker polypeptides are designed to be most suitable for the intended application, particularly in terms of length, flexibility, charge and solubility.

Variants of the chimeric fusion protein with one or several amino acid substitutions or deletions may also be used to obtain the recombinant DNA or chimeric fusion protein of the invention. Those skilled in the art can readily ascertain whether or not such variations of the procedures described in the examples section are suitable for the methods of the invention.

By replication in a suitable host cell, large quantities of the polynucleotide can be produced. Natural or synthetic DNA fragments encoding the proteins or fragments thereof will be incorporated into recombinant polynucleotide constructs, typically DNA constructs, capable of being introduced into and replicated in prokaryotic or eukaryotic cells.

Polynucleotides may also be produced by chemical synthesis, including but not limited to the phosphoramidite method described in Beaucage, S.L. and Caruthers, M.H., Tetrahedron Letters 22(1981) 1859-312 and the triester method according to Matteucci, M.D and Caruthers, M.H., J.Am.chem.Soc.103(1981) 3185-3191. Double-stranded fragments can be obtained from chemically synthesized single-stranded products by synthesizing complementary strands and annealing the strands together under suitable conditions or by adding complementary strands with a DNA polymerase and a suitable primer sequence.

Nucleotide sequences are operably linked when the polynucleotide sequence is placed in a functional relationship with another polynucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, not only contiguous but in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operably linked at a distance, even if not contiguous.

The DNA construct prepared for introduction into a host will typically contain a replication system recognized by the host, including the desired DNA segment encoding the desired chimeric fusion peptide and optionally additional target polypeptide, and will also preferably include transcriptional and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) can include, for example, an origin of replication or Autonomously Replicating Sequence (ARS) and expression control sequences, promoters, enhancers and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, transcription termination sequences and mRNA stabilizing sequences.

The appropriate promoter and other necessary vector sequences are selected to be functional in the host. Examples of cell lines that can work in combination with expression vectors include, but are not limited to, Sambrook, J. et al, Molecular Cloning: a Laboratory Manual "(1989) -, eds. J.Sambrook, E.F. Fritsch and T.Maniatis, Cold Spring harbor Laboratory Press, Cold Spring harbor Harbour, or Ausubel, F.et al in" Current protocols in molecular biology "(1987 and periodic updates), eds. F.Ausubel, R.Brent and K.R.E., Wiley & Sons Verlag, New York; and Metzger, D., et al, Nature 334(1988) 31-6. Many expression vectors useful in bacteria, yeast, mammalian, insect, plant or other cells are known in the art and are available from suppliers including, but not limited to, Stratagene, New England Biolabs, Promega Biotech, etc. In addition, the construct may be linked to an amplifiable gene (e.g., DHFE) such that multiple copies of the gene may be obtained.

Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector, although such a marker gene may be carried in another polynucleotide sequence co-introduced into the host cell. Only those host cells expressing the marker gene will survive and/or grow under the selection conditions. Typical selection genes include, but are not limited to, those encoding proteins that (a) confer resistance to antibiotics or other toxic substances, such as ampicillin. Tetracycline, etc.; (b) compensating for nutritional deficiencies; or (c) supplying key nutrients not available from complex media. The choice of the correct selectable marker will depend on the host cell, and suitable markers for different hosts are known in the art.

According to the present invention, it has proved very advantageous if an expression vector containing an operably linked recombinant DNA molecule according to the invention comprises a) at least one nucleotide sequence coding for a polypeptide binding segment of a non-human chaperone protein, b) upstream thereof at least one nucleotide sequence coding for an FK 506-binding protein or an FK 506-binding-protein-like domain (FKBP-like domain) and c) downstream thereof at least one nucleotide sequence coding for an FK 506-binding-protein-like domain (FKBP-like domain) and optionally d) at least one nucleotide sequence coding for a target polypeptide.

An expression vector comprising operably linked recombinant DNA molecules according to the invention, i.e. comprising a) at least one nucleotide sequence encoding a non-human chaperone polypeptide binding segment b) upstream at least one nucleotide sequence encoding a human FKBP-type peptidyl-proline cis-trans isomerase (PPIase) and, c) downstream at least one nucleotide sequence encoding a human FKBP-type peptidyl-proline cis-trans isomerase (PPIase), and optionally d) at least one nucleotide sequence encoding a target polypeptide, is also part of the invention.

The vector comprising the polynucleotide of interest can be introduced into the host cell by any method known in the art. These methods may vary depending on the type of cellular host, including but not limited to transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, other substances, and infection with viruses. A number of polynucleotides and polypeptides of the invention can be prepared by expressing a polynucleotide of the invention in a vector or other expression vehicle in a compatible host cell. The most commonly used prokaryotic host is an E.coli strain, although other prokaryotes, such as Bacillus subtilis, may also be used. Expression in E.coli represents a preferred mode of carrying out the invention.

The construction of the vectors according to the invention uses conventional ligation techniques. The isolated plasmids or DNA fragments are cut, trimmed and religated into the form required to produce the desired plasmid. If necessary, an analysis to confirm the correct sequence in the constructed plasmid is carried out in a known manner. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing assays to assess expression and function are known to those skilled in the art. Gene presence, amplification and/or expression can be measured directly in a sample by, for example, conventional southern blotting, northern blotting to quantify mRNA transcription, dot blotting (DNA or RNA analysis) or in situ hybridization using a suitably labeled probe that can be based on the sequences provided herein. One skilled in the art will readily envision how these methods may be modified if desired.

Expression vectors containing recombinant DNA according to the invention may be used for expressing fusion proteins in cell-free translation systems or for transforming host cells. In a preferred embodiment, the invention relates to a host cell transformed with an expression vector according to the invention.

In a further preferred embodiment, the invention relates to a method of producing a chimeric fusion protein. The method comprises the steps of culturing host cells transformed with the expression vector according to the invention, expressing the chimeric fusion protein in the respective host cells and purifying the chimeric fusion protein.

The chimeric fusion proteins according to the invention show high solubility. When overexpressed in the cytoplasm, they accumulate predominantly in the soluble fraction. To a lesser extent, they are also expressed in inclusion bodies. The cells are usually lysed under suitable buffer conditions, such as for example in a chaotropic substance. When the chimeric fusion protein is tagged with a hexahistidine moiety, unfolded proteins can be bound to a nickel-containing column (Ni-NTA), where they also refold under appropriate buffer conditions. Such purification and refolding procedures, as shown in more detail in the examples section, are well known to those skilled in the art. Due to its excellent folding helper properties, the chimeric fusion protein according to the invention can be used as a folding helper for any target protein that otherwise cannot adopt its correct three-dimensional structure, i.e. its native-like conformation. According to the invention, the chimeric fusion protein can also be used as a folding aid in a method for producing a target protein. For example, after initial lysis of an overproducing host cell, the overexpressed target protein does not usually adopt its native structure due to chaotropic substances or due to the presence of detergents or other buffering conditions, which threaten the native conformational state of the target protein. Chimeric fusion proteins may then be added during purification and solubilization of the target protein, and they may aid in the refolding and renaturation process.

For use in a coupled transcription/translation system, cell lysates containing over-expressed chimeric folding helpers can be added to vials in which in vitro translation is performed, thereby promoting proper conformational folding of the translated protein.

The chimeric fusion proteins according to the invention can be applied in immunoassays to help in the immunological binding process of antigens and antibodies to their binding partners without interfering with the immunoassay and its results. It is advantageous that the chimeric fusion proteins according to the invention are humanized, i.e. they comprise mainly human amino acid sequences, so that the possibility of interference due to naturally occurring antibodies against non-human protein sequences in human samples is minimized. Preferably, the percentage of amino acids derived from the human sequence is at least 60% compared to the complete amino acid sequence of the chimeric fusion protein.

Immunoassays are well known to those skilled in the art. The methods of carrying out such assays, as well as the practical applications and procedures are summarized in the relevant textbooks. Examples of relevant textbooks are Tij ssen, P., Preparation of enzyme-antibody or other enzyme-macromolecular conjugate "Practice and the term of enzyme immunological systems" (1990)221- "278, Eds.R.H.Burdon and v.P.H.Knippenberg, Elsevier, Amsterdam) and Tijssen," Methods in Enzymology "(1980), Eds.S.P.Colowick, N.O.Caplan and S.P., Academic Press), which relate to immunoassay Methods, in particular, volumes 70, 73, 74, 84, 92 and 121.

In a further embodiment of the invention, the chimeric fusion protein may be used as a fusion partner in a target protein production process, in a vaccine production process or in a method for producing a pharmaceutical, respectively.

For situations where the novel chimeric fusion protein is intended for therapeutic use, it is preferred to formulate a composition comprising the recombinantly produced chimeric fusion protein according to the invention and a pharmaceutically acceptable excipient.

The following examples, references, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made to the procedure described without departing from the spirit of the invention.

Examples

Materials andreagent

Guanidine chloride (GdmCl, grade a) was purchased from NIGU (Waldkraiburg, germany).EDTA-free protease inhibitor tablets, imidazole and EDTA were obtained from Roche diagnostics GmbH (Mannheim, Germany) and all other chemicals were of analytical grade from Merck (Darmstadt, Germany). (S54G, P55N) -RNase T1 was purified, reduced and carboxymethylated as described in Mulke, M. and Schmid, F.X. (1994) J.mol.biol.239, 713-. Ultrafiltration membranes (YM10, YM30) were purchased from Amicon (Danvers, MA, USA), microdialysis membranes (VS/0.025 μm) and ultrafiltration units (biomax ultra filters) from Millipore (Bedford, MA, USA). Nitrocellulose and cellulose acetate membranes (1.2 μm/0.45 μm/0.2 μm) for filtration of crude zymolyte were obtained from Sartorius (R) ((R))Germany).

Example 1

Production of chimeric fusion protein hFKBP12-IF1 comprising E.coli SlyD and human FKBP12 sequences

Cloning of the expression cassette

hFKBP12 and SlyD sequences were retrieved from the SwissProt database. Synthetic genes encoding hFKBP12 and its insertion variants were purchased from mediganomix (Martinsried, germany) and cloned into pET24 expression vector (Novagen, Madison, Wisconsin, USA). Codon usage was optimized for expression in E.coli host cells. The SlyD gene was PCR-amplified from E.coli strain BL21(DE3), restricted and ligated into pET24a expression vector. Coli SlyD as described in Scholz et al (2005) in J.mol.biol.345, 1229-^*The fusion module is used for designing an expression cassette of the fusion protein.

Encoding the protein FKBP12-IF1 (also shown in SEQ ID NO.10 with a six histidine tag)

MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFM

LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGQYDENL VQRVPKDVFM

GVDELQVGMR FLAETDQGPV PVEITAVEDD HVVVDGNHML AGQNLVFDVE

LLKLE

The synthetic gene of (2) was purchased from Medigenomix (Martinsried, Germany) and cloned into pET24a expression vector (Novagen, Madison, Wis.). Codon usage was optimized for expression in E.coli host cells. QuikChange (Stratagene, La Jolla, Calif.) was used to generate cysteine-free variants (C22A). The N-terminal methionine is removed post-translationally by cleavage with the bacterial N-methionyl-aminopeptidase, whereby the mature polypeptide is actually initiated with glycine 1.

To obtain the FKBP12-IF1(C22A) -gp41 fusion construct (also shown in SEQ ID NO.11),

MGVQVETISP GDGRTFPKRG QTAVVHYTGM LEDGKKFDSS RDRNKPFKFM

LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGQYDENL VQRVPKDVFM

GVDELQVGMR FLAETDQGPV PVEITAVEDD HVVVDGNHML AGQNLVFDVE

LLKLEGGGSG GGSGGGSGGG SGGGSGGGTL TVQARQLLSG IVQQQNNELR

AIEAQQHLEQ LTVWGTKQLQ ARELAVERYL KDQQLLGIWG CSGKLICTTA

VPWNASWSNK SLEQIWNNMT WMEWDREINN YTSLIHSLIE ESQNQQEKNE

QELLELDKWA SLWNWFNITN WLWYLEHHHH HH

PCR amplification of the encoded NdeI/BamHI-flanked FKBP12-IF1- (GGGS)₂GG and BamHI/XhoI-flanked (GGGS)₂A DNA fragment of GG-gp41 (residues 536 to 681) and inserted into pET24a with NdeI and XhoI. Synthetic gene encoding FKBP12-IF1(C22A)Or purified HIV-1 isolate RNA was used as PCR- (RT-PCR) -template. QuikChange was used to introduce point mutations L555E, L566E, I573T and I580E into the gp41 cassette.

Encoding BamHI/BamHI-flanked (GGGS) by cleavage of FKBP12-IF1(C22A) -gp41 with BamHI and insertion of PCR-amplified synthetic gene encoding FKBP12-IF1(C22A)₂GGG-F12IF1-(GGGS)₂GG, to generate a fusion construct (SEQ ID NO.7) of the tandem FKBP12-IF1(C22A) -FKBP12-IF1(C22A) -gp 41.

MGVQVETISP GDGRTFPKRG QTAVVHYTGM LEDGKKFDSS RDRNKPFKFM

LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGQYDENL VQRVPKDVFM

GVDELQVGMR FLAETDQGPV PVEITAVEDD HVVVDGNHML AGQNLVFDVE

LLKLEGGGSG GGSGGGSGGG SGGGSGGGGV QVETISPGDG RTFPKRGQTA

VVHYTGMLED GKKFDSSRDR NKPFKFMLGK QEVIRGWEEG VAQMSVGQRA

KLTISPDYAY GQYDENLVQR VPKDVFMGVD ELQVGMRFLA ETDQGPVPVE

ITAVEDDHVV VDGNHMLAGQ NLVFDVELLK LEGGGSGGGS GGGSGGGSGG

GSGGGTLTVQ ARQLLSGIVQ QQNNELRAIE AQQHLEQLTV WGTKQLQARE

LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLE QIWNNMTWME

WDREINNYTS LIHSLIEESQ NQQEKNEQEL LELDKWASLW NWFNITNWLW

YLEHHHHHH

EcSlyD-[GGGS]₅GGG-EcSlyD-[GGGS]₅GGG-gp41(536-681；L555E，L566E，I573T，I580E)-HGHDHDHD-His6，pET24a

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE

GHEVGDKFDV AVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET

DQGPVPVEIT AVEDDHVVVD GNHMLAGQNL KFNVEVVAIR EATEEELAHG

HVHGAHDHHH DHDHDGGGSG GGSGGGSGGG SGGGSGGGKV AKDLVVSLAY

QVRTEDGVLV DESPVSAPLD YLHGHGSLIS GLETALEGHE VGDKFDVAVG

ANDAYGQYDE NLVQRVPKDV FMGVDELQVG MRFLAETDQG PVPVEITAVE

DDHVVVDGNH MLAGQNLKFN VEVVAIREAT EEELAHGHVH GAHDHHHDHD

HDGGGSGGGS GGGSGGGSGG GSGGGTLTVQ ARQLLSGIVQ QQNNELRAIE

AQQHLEQLTV WGTKQLQARE LAVERYLKDQ QLLGIWGCSG KLICTTAVPW

NASWSNKSLE QIWNNMTWME WDREINNYTS LIHSLIEESQ NQQEKNEQEL

LELDKWASLW NWFNITNWLW YHGHDHDHDH HHHHH

Point mutations, deletions and insertions variants or restriction sites were generated using QuikChange (Stratagene, La Jolla, USA) and standard PCR techniques. All recombinant hFKBP12 variants contained a C-terminal hexahistidine tag to facilitate Ni-NTA-assisted purification and refolding.

Expression, purification and refolding of hFKBP12 variants

All hFKBP12, SlyD and SlpA variants as well as fusion proteins were purified using essentially the same protocol. Coli BL21(DE3) cells containing the specific pET24a expression plasmid were grown to OD at 37 ℃ on LB medium supplemented with kanamycin (30. mu.g/ml)₆₀₀1.5, and inducing overexpression of the cytoplasm by adding 1mM isopropyl-beta-D-thiogalactoside. Three hours after induction, cells were harvested by centrifugation (20 min at 5000 g), frozen and stored at-20 ℃. For cell lysis, the frozen pellet was resuspended in cold 50mM sodium phosphate (pH8.0), 7.0MGdmCl, 5mM imidazole, and the suspension was stirred on ice2 hours until complete cell lysis. After centrifugation and filtration (nitrocellulose membrane, 0.45 μm/0.2 μm), the lysate was added to a Ni-NTA column equilibrated with lysis buffer containing 5.0mM TCEP. The subsequent washing steps were adjusted for each target protein and ranged from 5-15mM imidazole in 50mM sodium phosphate (pH8.0), 7.0M GdmCl, 5.0mM TCEP. At least 10-15 volumes of wash buffer are used. Then, the GdmCl solution was replaced with 50mM sodium phosphate (pH7.8), 100mM NaCl, 10mM imidazole, 5.0mM TCEP to induce conformational refolding of the matrix-bound protein. To avoid co-purified protease reactivation, a mixture of protease inhibitors is included in the refolding buffer: (EDTA free, Roche). A total amount of 15-20 column volumes of refolding buffer was used in the overnight reaction. Then, TCEP was removed by washing with 3-5 column volumes of 50mM sodium phosphate (pH7.8), 100mM NaCl, 10mM imidazole andno EDTA inhibitor mixture. The native protein was then eluted with 250mM imidazole in the same buffer. By reacting N- [ 2-hydroxy-1, 1-dimethylol ethyl]The purity of the protein-containing fractions was estimated by glycine (Tricine) -SDS-PAGE and pooled. Finally, the proteins were size exclusion chromatographed (SuperdexHiLoad, Amersham Pharmacia) and the protein containing fractions were pooled and concentrated in an Amicon cell (YM 10).

After coupled purification and refolding procedures, greater than 20mg of target protein can be obtained from 1g of E.coli wet cells. We also improved the overall solubility of the various hFKBP12 variants by changing cysteine 22 to alanine. This substitution of a single cysteine abolished the tendency of hFKBP12 to form covalent disulfide adducts. This does not affect the folding of the protein nor its prolyl isomerase activity. Cysteine 22 substitution of alanine has also been shown to be beneficial for the chimeric protein FKBP12-IF 1. The purification of hFKBP12-IF1(C22A) as demonstrated by SDS-PAGE is shown in FIG. 1.

Examples2

Spectral measurement

Protein concentration measurements were performed with an Uvikon XL dual beam spectrophotometer. The molar extinction coefficient (. epsilon.) was determined using the procedure described by Pace (1995), Protein Sci.4, 2411-2423₂₈₀)。

near-UV circular dichroism spectra were recorded with a Jasco-720 optical rotation spectrometer with a thermostated cell holder and converted into mean residual ellipticity. The buffer solution was 50mM sodium phosphate (pH7.5), 100mM NaCl, 1mM EDTA. The optical path length is 0.5cm or 1.0cm, and the protein concentration is 20-500. mu.M. The bandwidth was 1nm, the scanning speed was 20 nm/min, the resolution was 0.5nm, and the response was 2 seconds. To improve the signal-to-noise ratio, the spectra were measured nine times and averaged.

Evaluation of native-like folding by near ultraviolet CD

To examine whether the chimeric fusion proteins of the invention adopt a folded conformation after coupled purification and refolding procedures, we measured circular dichroism spectra in the near ultraviolet region. Near ultraviolet CD reports the asymmetric environment of aromatic residues in proteins and is therefore a sensitive test for ordered tertiary structure. Grafting of domains such as the SlyD IF loop into the hFKBP12flap segment may seriously compromise the overall structure of hFKBP12 scaffold protein. Natural human FKBP12 has typical CD characteristics in the near ultraviolet region (fig. 2). Therefore, structural deformations or collisions due to IF-ring-insertion should be visible in the near-ultraviolet circular dichroism spectrum. FIG. 2 shows the spectral coverage of hFKBP12 and hFKBP12-IF1, respectively. Surprisingly, the insertion of the IF domain in the flap region of hFKBP12 maintained the scaffold protein overall structure essentially intact. The spectral characteristics are similar even though the ellipticity of the transition is in fact significantly reduced due to the ring insertion. The results strongly suggest that the native-like folding of the chimeric construct hFKBP12-IF1 is essentially preserved, since global unfolding will abolish any near-uv CD signal.

Similarly, we recorded SlyD^*(SlyD 1-165) and deletion variants thereofBody SlyD^*Delta IF-Ring (SlyD)^*Lacking amino acid residues 70-129). These results are shown in FIG. 3. SlyD when large "insert in flap" (IF) domains are removed, as judged by near-UV circular dichroism^*The overall structure of (a) remains intact. The ovality of the average residue transition even slightly increased after removal of the IF loop. Thus, there is strong evidence that SlyD lacking its IF domain is still a naturally-like folded protein.

Example 3

Folding experiment

For the folding studies, the reduced and carboxymethylated RNase T1(RCM-T1) was used. RCM-T1 was unfolded by incubating the protein in 0.1M Tris-HCl pH8.0 for at least 1 hour at 15 ℃. Refolding was initiated by 40-fold dilution of the unfolded protein to final conditions of 2.0M NaCl and desired concentrations of SlyD, FKBP12 variant and RCM-T1 in the same buffer at 15 ℃. After the folding reaction, the protein fluorescence (i.e., tryptophan fluorescence) at 320nm (10nm bandwidth) was enhanced after 268nm (1.5nm bandwidth) excitation. The slow folding of RCM-T1 at 2.0M NaCl is a single exponential process and its rate constants are determined using the program GraFit 3.0 (Erithocus Software, Staines, UK).

Folding Activity of chimeric fusion proteins

We investigated the efficiency of the chimeric fusion proteins according to the invention in catalyzing proline-limited protein folding reactions. Reduced and carboxymethylated RNase T1(RCM-T1) was used as a model substrate. The refolding reaction is accompanied by a strong increase in tryptophan fluorescence and can be induced by increasing the concentration of NaCl as described by Schmid, F.X, (1991) Curr, Opin, Struct, biol.1, 36-41, Mayr et al (1996) Biochemistry 35, 5550-.

SlyD from E.coli^*(1-165) very well catalyses the refolding of RCM-T1. SlyD at as low as 2nM^*Re-folding of RCM-T1 in the Presence ofThe stack has accelerated twice (fig. 4A). Apparent first order folding rate constant k_appFollowed by SlyD^*The concentration increased linearly (fig. 4B). From the slope of the plot, the specificity constant k is determined_cat/K_MIs 0.68X 10⁶M^-1s^-1. This is an exceptionally high value, which almost achieves the catalytic efficiency of the fold replicon initiating factor known to date to be most effective (see Stoller et al (1995) EMBO J.14, 4939-.

In contrast, SlyD mutants lacking the IF domain are very weak catalysts for RCM-T1 refolding. SlyD. DELTA.IF represents the FKBP-domain of SlyD. The specificity constant range is 0.0005 multiplied by 10⁶M^-1s^-1Left and right and thus equal to SlyD^*Only 0.07% (fig. 5A/B). This strongly indicates the important role of the "insert in flap" domain for binding to unfolded protein substrates. Since the near-uv circular dichroism spectrum points to the natural-like global folding of the deletion variants (fig. 3), the insert in flap domain may represent the polypeptide-binding domain, i.e. the partner domain of SlyD.

Deletion of the putative IF Domain virtually abolished SlyD^*Folding activity of (a). SlyD mutants lacking the IF domain represent the FKBP or FKBP-like domain of SlyD. In contrast, how does one influence the folding activity of hFKBP12 by inserting exactly this IF element into the flap region? In agreement with published data (Scholz et al (1997) EMBO J.16, 54-58), hFKBP12 catalyzes RCM-T1 refolding quite mildly (FIG. 6A). Analysis of the apparent first-order refolding rate constant gave 0.014X 10⁶M^-1s^-1The specificity constant (fig. 6B). In contrast, the IF-loop insertion variant FKBP12-IF1 according to the invention catalyses RCM-T1 refolding very well (FIG. 7A). FKBP12-IF1 below 1nM was sufficient to double the folding rate of RCM-T1. The specificity constant is higher than 2.5X 10⁶M^-1s^-1(FIG. 7B and Table 1). This outstanding value even exceeds the catalytic efficiency of the initiation factor, which is equal to 1.2X 10⁶M^-1s^-1(Stoller et al (1995)EMBO J.14, 4939-; zarnt et al (1997) J.mol.biol.271, 827-837; scholz et al (1997) EMBO J.16, 54-58). Thus, by constructing the chimeric fusion proteins of the invention, we converted mild non-human prolyl isomerase and weak human chaperones into superior folding helpers with exceptionally good prolyl isomerase and chaperone properties.

The principle of combining the polypeptide binding domain of a non-human chaperone with the human PPIase domain according to the present invention can be extended to other examples. Similar to the construction pattern of FKBP12-IF1, we grafted the putative SlpA IF loop domain onto the hFKBP12 folding scaffold. SlpA (acronym stands for SlyD-like protein A) is closely related to SlyD. Information on SlpA is rare, but due to its homology to SlyD, its role as a PPIase chaperone in the escherichia coli cytoplasm is presumed. We purified and characterized the hexahistidine-tagged SlpA variant from e. However, this putative PPIase showed very weak activity in the RCM-T1 refolding assay (Table 1). The chimera containing elements from hFKBP12 and SlpA was called hFKBP12-IF 4. It contains the modules G1-G83 from hFKBP12, V72-T132 from SlpA and L97-E107 from hFKBP12 (sequence information see SEQ ID NO. 13). Expression, purification to homogeneity and refolding of the hexahistidine-tagged protein were accomplished essentially as described for FKBP12-IF 1. The spectrum clearly pointing to the compact fold of the designed protein was obtained by near uv CD evaluation (spectrum not shown).

When evaluated in the RCM-T1 refolding assay, hFKBP12-IF4 exhibited surprisingly high folding activity (FIG. 10A). Its specificity constant (k)_cat/K_M) Is 800,000M^-1s^-1(see table 1) and in fact equals the catalytic efficiency of SlyD, which is a very efficient folding aid (Scholz et al, Biochemistry 2006, 45, 20-33). Again, the putative polypeptide binding domain (from SlpA) in combination with a tardive prolyl isomerase (hFKBP12) yields a superior folding helper with high enzymatic and chaperone activity. We can conclude that combining hFKBP12 with IP loop domains from different SlyD homologues can result in a polypeptide with a desired binding specificityA humanized folding helper for aberrant folding activity.

Another example of a principle of combining the polypeptide binding domain of a non-human chaperone with a human PPIase domain is the chimeric fusion protein known as hFKBP12-IF 5. Based on the construction patterns of FKBP12-IF1 and FKBP12-IF4, we grafted the putative IF loop domain of Thermococcus FKBP18 onto the folding scaffold of hFKBP 12. Pyrococcus FKBP18 is a thermostable homologue of SlyD with a putative IF domain near the prolyl isomerase active site in the flap region.

The resulting chimera was designated hFKBP12-IF 5. It contains the module G1-G83 from hFKBP12, M84-T140 from Pyrococcus FKBP18 and L97-E107 from hFKBP12 (sequence information for Pyrococcus FKBP18 is shown in SEQ ID NO.14 and for hFKBP12-IF5 is shown in SEQ ID NO. 15). Expression, purification to homogeneity and refolding of the hexahistidine-tagged protein were accomplished essentially as described for FKBP12-IF 1.

When evaluated in the RCM-T1 refolding assay, hFKBP12-IF5 exhibited surprisingly high folding activity (FIG. 11). Its specificity constant (k)_cat/K_M) Is 660,000M^-1s^-1And in fact equal to the catalytic efficiency of SlyD, the latter is a very effective folding aid according to recent literature data (Scholz et al, Biochemistry 2006, 45, 20-33). Again, the putative polypeptide binding domain (from thermostable TcFKBP18) in combination with a sluggish prolyl isomerase (hFKBP12) produced superior folding helpers with high enzymatic and chaperone activity. Our studies clearly show that the combination of hFKBP12 with IP loop domains from different SlyD homologues results in humanized folding helpers with aberrant folding activity.

It is this same principle that also applies to FKBP-like domains present in many prokaryotic and eukaryotic prolyl isomerases. For example, FkpA and the priming factor are two E.coli proteins containing FKBP-like domains. It has previously been shown that these FKBP-like domains, when separated from the rest of the molecule, exhibit very mild folding activity (Scholz et al, EMBO J. (1997)16(1) 54-58; Saul et al, J.mol.biol. (2004)335, 595-608). This is in perfect agreement with human FKBP12, a mild prolyl isomerase that lacks any chaperone activity. Excellent folding aids can be obtained by grafting any SlyD IF domain (also called polypeptide binding segment) onto the FKBP-like domain as folding scaffold. These chimeras can be used as folding aids in recombinant protein biotechnology, for example as fusion proteins, as additives in refolding buffers, etc.

The principle of domain grafting is also applicable to SlyD itself. SlyD deletion variants lacking the IF domain (SlyD Δ IF) represent authentic FKBP domains that can be combined with IF domains from other FKBP chaperones to produce folding helpers with exceptional catalytic efficiency. The invention therefore includes the use of SlyD, in particular SlyD variants lacking an IF domain, for the production of a folding helper having a catalytic efficiency that exceeds that of the naturally occurring wild-type molecule used for the preparation of the resulting chimeric folding helper.

Table 1 summarizes the results obtained for all proteins measured in the RCM-T1 assay.

TABLE 1

PPIase variants	Specificity constant kcat/KM(M-1s-1)
		hFKBP12	14,000
hFKBP12(C22A)	14,000
		SlyD＊(SlyD 1-165)	680,000
SlyD＊Delta IF ring	500
		hFKBP12-IF1 (C22A)/invention	2,500,000
SlpA	＜1000
		hFKBP12-IF 4/invention	850,000
TcFKBP18	600,000
		hFKBP12-IF 5/invention	660,000

Example 4

Immunoreactivity of FKBP12-IF1/HIV gp41 fusion protein

This example shows that the chimeric fusion protein FKBP12-IF1 of the invention is supplemented with the HIV protein, i.e. the envelope protein gp41, as target protein. The tandem FKBP12-IF1-gp41 fusion module was purified and refolded as described for the SlyD and FKBP12 protein variants. It was used to detect a number of anti-gp 41 antibodies present in HIV-1 positive sera. Using a double antigen sandwich format in automationImmunoreactivity was challenged in a 2010 analyser (Roche Diagnostics GmbH, Germany).

In thatSignal detection in immunoassays is based on electrochemiluminescence. The biotin conjugate (i.e., capture antigen) is immobilized on streptavidin-coated magnetic beads, while the signaling antigen carries a complexed ruthenium cation as the light-emitting moiety. In the presence of anti-gp 41 antibody, the chromogenic ruthenium complex bridged to the solid phase and emitted light at 620nm after excitation with a platinum electrode. The signal output is an arbitrary light unit.

For them asUse of the antigen, concentration of the soluble gp41 fusion protein under investigation and modification with N-hydroxy-succinimide activated biotin and ruthenium moieties as described by Scholz et al (2005) J.mol.biol.345, 1229-1241. The gp41 variant was present at a concentration of about 500ng/ml in the immunoassay measurement. At least five negative sera were used as controls. To further minimize false positive results, polymerized unlabeled E.coli SlyD was added to the samples^*As an anti-interference substance.

The results show that the chimeric fusion FKBP12-IF1 according to the invention is very suitable as fusion partner for proteins with a tendency to aggregate. When wild-type hFKBP12 was fused to a gp41 ectodomain fragment, the resulting fusion protein quantitatively aggregated after matrix-coupled refolding and imidazole elution. Apparently, hFKBP12 was unable to confer solubility on extremely hydrophobic targets such as gp41 ectodomain fragments. In contrast, we found that the chimeric fusion protein according to the invention containing FKBP12-IF1 and HIV-1gp41 ectodomain fragment 536-681 (scheme 8) is very soluble and does not tend to aggregate as judged by UV spectroscopy (FIG. 9). When in automatic ElecsysAnalyzerAt the time of middle evaluation, the results demonstrated that they are well suited for diagnostic applications in HIV-1 serology (data not shown). This further highlights the outstanding properties of the chimeric fusion proteins according to the invention.

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Sequence listing

<110>Roche-Diagnostics GmbH；F.Hoffmann-La Roche AG

<120> chimeric fusion protein having excellent chaperone and folding activities

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Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg

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Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu

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Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala

35 40 45

Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala

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Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro

65 70 75 80

Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe

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Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val

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Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln

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Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu

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Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His

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Asp His Asp His Asp Gly Cys Cys Gly Gly His Gly His Asp His Gly

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His Glu His Gly Gly Glu Gly Cys Cys Gly Gly Lys Gly Asn Gly Gly

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Cys Gly Cys His

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Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

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Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

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Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala

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Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

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Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu

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Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

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Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

35 40 45

Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala

50 55 60

Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

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Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr

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Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu His His His His His

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His

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<213> artificial; hFKPB12-IF1 with SlyD insert

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Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

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Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp

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Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

35 40 45

Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala

50 55 60

Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

65 70 75 80

Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp

85 90 95

Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala

100 105 110

Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp

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Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu

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Val Phe Asp Val Glu Leu Leu Lys Leu Glu

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<210>5

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Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

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Lys Arg Gly Gln Thr Ala Val Val His Tyr Thr Gly Met Leu Glu Asp

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Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

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Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala

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Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

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Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp

85 90 95

Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala

100 105 110

Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp

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Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu

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Val Phe Asp Val Glu Leu Leu Lys Leu Glu

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<210>6

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<212>PRT

<213> artificial; SlyD-HIV gp41 fusion protein

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Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg

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Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu

20 25 30

Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala

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Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala

50 55 60

Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro

65 70 75 80

Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe

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Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val

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Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln

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Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu

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Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His

145 150 155 160

Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

165 170 175

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Val Ala Lys

180 185 190

Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg Thr Glu Asp Gly Val

195 200 205

Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu Asp Tyr Leu His Gly

210 215 220

His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala Leu Glu Gly His Glu

225 230 235 240

Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala Asn Asp Ala Tyr Gly

245 250 255

Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met

260 265 270

Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp

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Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val

290 295 300

Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Lys Phe Asn

305 310 315 320

Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu Glu Glu Leu Ala His

325 330 335

Gly His Val His Gly Ala His Asp His His His Asp His Asp His Asp

340 345 350

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

355 360 365

Gly Gly Gly Ser Gly Gly Gly Thr Leu Thr Val Gln Ala Arg Gln Leu

370 375 380

Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Glu Leu Arg Ala Ile Glu

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Ala Gln Gln His Leu Glu Gln Leu Thr Val Trp Gly Thr Lys Gln Leu

405 410 415

Gln Ala Arg Glu Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu

420 425 430

Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val

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Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn

485 490 495

Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp

500 505 510

Phe Asn Ile Thr Asn Trp Leu Trp Tyr His Gly His Asp His Asp His

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Asp His His His His His His

530 535

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<213> artificial; hFKBP12-IF1-hFKBP12-IF1-gp41 fusion protein

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Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

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Pro Lys Arg Gly Gln Thr Ala Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys

85 90 95

Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu

100 105 110

Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu

115 120 125

Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn

130 135 140

Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

165 170 175

Gly Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr

180 185 190

Phe Pro Lys Arg Gly Gln Thr Ala Val Val His Tyr Thr Gly Met Leu

195 200 205

Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe

210 215 220

Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly

225 230 235 240

Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro

245 250 255

Asp Tyr Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro

260 265 270

Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe

275 280 285

Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val

290 295 300

Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln

305 310 315 320

Asn Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Gly Ser

325 330 335

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

340 345 350

Gly Gly Gly Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile

355 360 365

Val Gln Gln Gln Asn Asn Glu Leu Arg Ala Ile Glu Ala Gln Gln His

370 375 380

Leu Glu Gln Leu Thr Val Trp Gly Thr Lys Gln Leu Gln Ala Arg Glu

385 390 395 400

Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp

405 410 415

Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala

420 425 430

Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met Thr Trp

435 440 445

Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser

450 455 460

Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu

465 470 475 480

Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr

485 490 495

Asn Trp Leu Trp Tyr Leu Glu His His His His His His

500 505

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<213> artificial; SlyD with six-His tag (SlyD 1-165)

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Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg

1 5 10 15

Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu

20 25 30

Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala

35 40 45

Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala

50 55 60

Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro

65 70 75 80

Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe

85 90 95

Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val

100 105 110

Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln

115 120 125

Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu

130 135 140

Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His

145 150 155 160

Asp His Asp His Asp His His His His His His

165 170

<210>9

<211>124

<212>PRT

<213> artificial; SlyD without IF-ring (SlyD 1-165)

<400>9

Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg

1 5 10 15

Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu

20 25 30

Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala

35 40 45

Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala

50 55 60

Asn Asp Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His

65 70 75 80

Ala Thr Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr

85 90 95

Glu Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His

100 105 110

His Asp His Asp His Asp His His His His His His

115 120

<210>10

<211>161

<212>PRT

<213> artificial; hFKBP12-IF1 with six-His tag

<400>10

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys

85 90 95

Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu

100 105 110

Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu

115 120 125

Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn

130 135 140

Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu His His His His His

145 150 155 160

His

<210>11

<211>332

<212>PRT

<213> artificial; hFKBP12-IF1(C22A) -gp41 fusion protein

<400>11

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Ala Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys

85 90 95

Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu

100 105 110

Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu

115 120 125

Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn

130 135 140

Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

165 170 175

Gly Gly Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val

180 185 190

Gln Gln Gln Asn Asn Glu Leu Arg Ala Ile Glu Ala Gln Gln His Leu

195 200 205

Glu Gln Leu Thr Val Trp Gly Thr Lys Gln Leu Gln Ala Arg Glu Leu

210 215 220

Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly

225 230 235 240

Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser

245 250 255

Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met Thr Trp Met

260 265 270

Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu

275 280 285

Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu

290 295 300

Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn

305 310 315 320

Trp Leu Trp Tyr Leu Glu His His His His His His

325 330

<210>12

<211>148

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>12

Ser Glu Ser Val Gln Ser Asn Ser Ala Val Leu Val His Phe Thr Leu

1 5 10 15

Lys Leu Asp Asp Gly Thr Thr Ala Glu Ser Thr Arg Asn Asn Gly Lys

20 25 30

Pro Ala Leu Phe Arg Leu Gly Asp Ala Ser Leu Ser Glu Gly Leu Glu

35 40 45

Gln His Leu Leu Gly Leu Lys Val Gly Asp Lys Thr Thr Phe Ser Leu

50 55 60

Glu Pro Asp Ala Ala Phe Gly Val Pro Ser Pro Asp Leu Ile Gln Tyr

65 70 75 80

Phe Ser Arg Arg Glu Phe Met Asp Ala Gly Glu Pro Glu Ile Gly Ala

85 90 95

Ile Met Leu Phe Thr Ala Met Asp Gly Ser Glu Met Pro Gly Val Ile

100 105 110

Arg Glu Ile Asn Gly Asp Ser Ile Thr Val Asp Phe Asn His Pro Leu

115 120 125

Ala Gly Gln Thr Val His Phe Asp Ile Glu Val Leu Glu Ile Asp Pro

130 135 140

Ala Leu Glu Ala

145

<210>13

<211>161

<212>PRT

<213> artificial; hFKBP12-IF4(SlpA insert) with six-His tag

<400>13

Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

1 5 10 15

Lys Arg Gly Gln Thr Ala Val Val His Tyr Thr Gly Met Leu Glu Asp

20 25 30

Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

35 40 45

Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala

50 55 60

Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

65 70 75 80

Ala Tyr Gly Val Pro Ser Pro Asp Leu Ile Gln Tyr Phe Ser Arg Arg

85 90 95

Glu Phe Met Asp Ala Gly Glu Pro Glu Ile Gly Ala Ile Met Leu Phe

100 105 110

Thr Ala Met Asp Gly Ser Glu Met Pro Gly Val Ile Arg Glu Ile Asn

115 120 125

Gly Asp Ser Ile Thr ValAsp Phe Asn His Pro Leu Ala Gly Gln Thr

130 135 140

Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu His His His His His

145 150 155 160

His

<210>14

<211>159

<212>PRT

<213> Thermococcus sp

<400>14

Met Lys Val Glu Ala Gly Asp Tyr Val Leu Phe His Tyr Val Gly Arg

1 5 10 15

Phe Glu Asp Gly Glu Val Phe Asp Thr Ser Tyr Glu Glu Ile Ala Arg

20 25 30

Glu Asn Gly Ile Leu Val Glu Glu Arg Glu Tyr Gly Pro Met Trp Val

35 40 45

Arg Ile Gly Val Gly Glu Ile Ile Pro Gly Leu Asp Glu Ala Ile Ile

50 55 60

Gly Met Glu Ala Gly Glu Lys Lys Thr Val Thr Val Pro Pro Glu Lys

65 70 75 80

Ala Tyr Gly Met Pro Asn Pro Glu Leu Val Ile Ser Val Pro Arg Glu

85 90 95

Glu Phe Thr Lys Ala Gly Leu Glu Pro Gln Glu Gly Leu Tyr Val Met

100 105 110

Thr Asp Ser Gly Ile Ala Lys Ile Val Ser Val Gly Glu Ser Glu Val

115 120 125

Ser Leu Asp Phe Asn His Pro Leu Ala Gly Lys Thr Leu Val Phe Glu

130 135 140

Val Glu Val Ile Glu Val Lys Lys Ala Glu Glu Asp Ser Glu Ala

145 150 155

<210>15

<211>157

<212>PRT

<213> artificial; hFKBP12-IF5 Thermococcus insert

<400>15

Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

1 5 10 15

Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp

20 25 30

Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

35 40 45

Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala

50 55 60

Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

65 70 75 80

Ala Tyr Gly Met Pro Asn Pro Glu Leu Val Ile Ser Val Pro Arg Glu

85 90 95

Glu Phe Thr Lys Ala Gly Leu Glu Pro Gln Glu Gly Leu Tyr Val Met

100 105 110

Thr Asp Ser Gly Ile Ala Lys Ile Val Ser Val Gly Glu Ser Glu Val

115 120 125

Ser Leu Asp Phe Asn His Pro Leu Ala Gly Lys Thr Leu Val Phe Asp

130 135 140

Val Glu Leu Leu Lys Leu Glu His His His His His His

145 150 155

<210>16

<211>432

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>16

Met Gln Val Ser Val Glu Thr Thr Gln Gly Leu Gly Arg Arg Val Thr

1 5 10 15

Ile Thr Ile Ala Ala Asp Ser Ile Glu Thr Ala Val Lys Ser Glu Leu

20 25 30

Val Asn Val Ala Lys Lys Val Arg Ile Asp Gly Phe Arg Lys Gly Lys

35 40 45

Val Pro Met Asn Ile Val Ala Gln Arg Tyr Gly Ala Ser Val Arg Gln

50 55 60

Asp Val Leu Gly Asp Leu Met Ser Arg Asn Phe Ile Asp Ala Ile Ile

65 70 75 80

Lys Glu Lys Ile Asn Pro Ala Gly Ala Pro Thr Tyr Val Pro Gly Glu

85 90 95

Tyr Lys Leu Gly Glu Asp Phe Thr Tyr Ser Val Glu Phe Glu Val Tyr

100 105 110

Pro Glu Val Glu Leu Gln Gly Leu Glu Ala Ile Glu Val Glu Lys Pro

115 120 125

Ile Val Glu Val Thr Asp Ala Asp Val Asp Gly Met Leu Asp Thr Leu

130 135 140

Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp Gly Ala Val Glu Ala

145 150 155 160

Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser Val Asp Gly Glu Glu

165 170 175

Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu Ala Met Gly Gln Gly

180 185 190

Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys Gly His Lys Ala Gly

195 200 205

Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu Glu Tyr His Ala Glu

210 215 220

Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile Asn Leu Lys Lys Val

225 230 235 240

Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu Phe Ile Lys Arg Phe

245 250 255

Gly Val Glu Asp Gly Ser Val Glu Gly Leu Arg Ala Glu Val Arg Lys

260 265 270

Asn Met Glu Arg Glu Leu Lys Ser Ala Ile Arg Asn Arg Val Lys Ser

275 280 285

Gln Ala Ile Glu Gly Leu Val Lys Ala Asn Asp Ile Asp Val Pro Ala

290 295 300

Ala Leu Ile Asp Ser Glu Ile Asp Val Leu Arg Arg Gln Ala Ala Gln

305 310 315 320

Arg Phe Gly Gly Asn Glu Lys Gln Ala Leu Glu Leu Pro Arg Glu Leu

325 330 335

Phe Glu Glu Gln Ala Lys Arg Arg Val Val Val Gly Leu Leu Leu Gly

340 345 350

Glu Val Ile Arg Thr Asn Glu Leu Lys Ala Asp Glu Glu Arg Val Lys

355 360 365

Gly Leu Ile Glu Glu Met Ala Ser Ala Tyr Glu Asp Pro Lys Glu Val

370 375 380

Ile Glu Phe Tyr Ser Lys Asn Lys Glu Leu Met Asp Asn Met Arg Asn

385 390 395 400

Val Ala Leu Glu Glu Gln Ala Val Glu Ala Val Leu Ala Lys Ala Lys

405 410 415

Val Thr Glu Lys Glu Thr Thr Phe Asn Glu Leu Met Asn Gln Gln Ala

420 425 430

<210>17

<211>112

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>17

Met Leu Asp Thr Leu Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp

1 5 10 15

Gly Ala Val Glu Ala Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser

20 25 30

Val Asp Gly Glu Glu Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu

35 40 45

Ala Met Gly Gln Gly Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys

50 55 60

Gly His Lys Ala Gly Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu

65 70 75 80

Glu Tyr His Ala Glu Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile

85 90 95

Asn Leu Lys Lys Val Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu

100 105 110

<210>18

<211>164

<212>PRT

<213> artificial; priming factor for IF inserts with SlyD

<400>18

Met Leu Asp Thr Leu Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp

1 5 10 15

Gly Ala Val Glu Ala Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser

20 25 30

Val Asp Gly Glu Glu Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu

35 40 45

Ala Met Gly Gln Gly Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys

50 55 60

Gly His Lys Ala Gly Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu

65 70 75 80

Glu Tyr His Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp

85 90 95

Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala

100 105 110

Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp

115 120 125

Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Ala

130 135 140

Lys Phe Ala Ile Asn Leu Lys Lys Val Glu Glu Arg Glu Leu Pro Glu

145 150 155 160

Leu Thr Ala Glu

<210>19

<211>270

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>19

Met Lys Ser Leu Phe Lys Val Thr Leu Leu Ala Thr Thr Met Ala Val

1 5 10 15

Ala Leu His Ala Pro Ile Thr Phe Ala Ala Glu Ala Ala Lys Pro Ala

20 25 30

Thr Ala Ala Asp Ser Lys Ala Ala Phe Lys Asn Asp Asp Gln Lys Ser

35 40 45

Ala Tyr Ala Leu Gly Ala Ser Leu Gly Arg Tyr Met Glu Asn Ser Leu

50 55 60

Lys Glu Gln Glu Lys Leu Gly Ile Lys Leu Asp Lys Asp Gln Leu Ile

65 70 75 80

Ala Gly Val Gln Asp Ala Phe Ala Asp Lys Ser Lys Leu Ser Asp Gln

85 90 95

Glu Ile Glu Gln Thr Leu Gln Ala Phe Glu Ala Arg Val Lys Ser Ser

100 105 110

Ala Gln Ala Lys Met Glu Lys Asp Ala Ala Asp Asn Glu Ala Lys Gly

115 120 125

Lys Glu Tyr Arg Glu Lys Phe Ala Lys Glu Lys Gly Val Lys Thr Ser

130 135 140

Ser Thr Gly Leu Val Tyr Gln Val Val Glu Ala Gly Lys Gly Glu Ala

145 150 155 160

Pro Lys Asp Ser Asp Thr Val Val Val Asn Tyr Lys Gly Thr Leu Ile

165 170 175

Asp Gly Lys Glu Phe Asp Asn Ser Tyr Thr Arg Gly Glu Pro Leu Ser

180 185 190

Phe Arg Leu Asp Gly Val Ile Pro Gly Trp Thr Glu Gly Leu Lys Asn

195 200 205

Ile Lys Lys Gly Gly Lys Ile Lys Leu Val Ile Pro Pro Glu Leu Ala

210 215 220

Tyr Gly Lys Ala Gly Val Pro Gly Ile Pro Pro Asn Ser Thr Leu Val

225 230 235 240

Phe Asp Val Glu Leu Leu Asp Val Lys Pro Ala Pro Lys Ala Asp Ala

245 250 255

Lys Pro Glu Ala Asp Ala Lys Ala Ala Asp Ser Ala Lys Lys

260 265 270

<210>20

<211>114

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>20

Gly Leu Val Tyr Gln Val Val Glu Ala Gly Lys Gly Glu Ala Pro Lys

1 5 10 15

Asp Ser Asp Thr Val Val Val Asn Tyr Lys Gly Thr Leu Ile Asp Gly

20 25 30

Lys Glu Phe Asp Asn Ser Tyr Thr Arg Gly Glu Pro Leu Ser Phe Arg

35 40 45

Leu Asp Gly Val Ile Pro Gly Trp Thr Glu Gly Leu Lys Asn Ile Lys

50 55 60

Lys Gly Gly Lys Ile Lys Leu Val Ile Pro Pro Glu Leu Ala Tyr Gly

65 70 75 80

Lys Ala Gly Val Pro Gly Ile Pro Pro Asn Ser Thr Leu Val Phe Asp

85 90 95

Val Glu Leu Leu Asp Val Lys Pro Ala Pro Leu Glu His His His His

100 105 110

His His

<210>21

<211>162

<212>PRT

<213> artificial; FkpA with IF-insert of SlyD

<400>21

Gly Leu Val Tyr Gln Val Val Glu Ala Gly Lys Gly Glu Ala Pro Lys

1 5 10 15

Asp Ser Asp Thr Val Val Val Asn Tyr Lys Gly Thr Leu Ile Asp Gly

20 25 30

Lys Glu Phe Asp Asn Ser Tyr Thr Arg Gly Glu Pro Leu Ser Phe Arg

35 40 45

Leu Asp Gly Val Ile Pro Gly Trp Thr Glu Gly Leu Lys Asn Ile Lys

50 55 60

Lys Gly Gly Lys Ile Lys Leu Val Ile Pro Pro Glu Leu Ala Tyr Gly

65 70 75 80

Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met

85 90 95

Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp

100 105 110

Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val

115 120 125

Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Val Phe Asp

130 135 140

Val Glu Leu Leu Asp Val Lys Pro Ala Pro Leu Glu His His His His

145 150 155 160

His His

Claims (15)

1. Comprises a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 4. SEQ ID NO: 13 or SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

2. The recombinant DNA molecule according to claim 1, characterized in that it further comprises

At least one nucleotide sequence encoding a target polypeptide.

3. An expression vector comprising an operably linked recombinant DNA molecule according to claim 1 or 2.

4. A host cell transformed with the expression vector according to claim 3.

5. A method of producing a chimeric fusion protein, the method comprising the steps of:

a) culturing the host cell according to claim 4

b) Expression of the chimeric fusion protein and

c) purification of the chimeric fusion protein.

6. A recombinantly produced chimeric fusion protein produced according to the method of claim 5.

7. A recombinantly produced chimeric fusion protein comprising a sequence according to SEQ ID NO: 4. SEQ ID NO: 13 or SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

8. Recombinantly produced fusion protein according to claim 7, characterized in that it further comprises at least one target polypeptide.

9. Use of a recombinantly produced chimeric fusion protein according to claims 6-8 for the preparation of an adjunct for immunoassays or a diagnostic kit.

10. Use of a recombinantly produced chimeric fusion protein according to claims 6-8 as a folding aid for a target protein.

11. Use of a recombinantly produced chimeric fusion protein according to claims 6-8 as a folding helper in a method of production of a target protein.

12. Use of a recombinantly produced chimeric fusion protein according to claims 6-8 as fusion partner in a method of production of a target protein.

13. Use of a recombinantly produced chimeric fusion protein according to claims 6-8 for the preparation of a kit for an immunoassay.

14. Use of a recombinantly produced chimeric fusion protein according to claims 6-8 in a method of producing a medicament.

15. A composition comprising a recombinantly produced chimeric fusion protein according to claims 6-8 and a pharmaceutically acceptable excipient.