CN104250288B - Amphiphilicα-helix self-assembling peptides and its application - Google Patents

Abstract

The present invention relates to an amphipathic α-helical self-assembling peptide derived from SEQ ID NO: 1 and its use in protein production and purification. When expressed in fusion with the target protein, the amphipathic α-helical self-assembling peptide can induce the fusion protein to form active aggregates in the cell, and compared with the polypeptide of SEQ ID NO: 1, the active aggregate formed has a higher concentration in the cell. The spatial distribution is altered, improving the host cell growth state and increasing the yield of the fusion protein.

Description

Amphiphilic α-helical self-assembling peptides and their applications

technical field

The invention relates to the field of genetic engineering. In particular, the present invention relates to novel amphipathic alpha-helical self-assembling peptides and their use in protein production and purification.

Background technique

With the increasing demand for protein and polypeptide products such as enzyme preparations and protein drugs, the use of prokaryotic systems to express recombinant proteins has become more and more widely used in industrial biotechnology. When expressing foreign genes in prokaryotic systems, the target protein tends to exist in the form of insoluble protein aggregates (also called inclusion bodies)[1]. Traditionally, inclusion bodies refer to insoluble precipitates formed by misfolding polypeptides that have no biological activity [2]. However, protein expression in the form of inclusion bodies has many advantages, such as large protein expression, high purity, easy separation and operation, avoiding protease degradation, etc., and is suitable for the production of exogenous proteins or polypeptides that are toxic to cells. However, the proteins expressed in the form of inclusion bodies need to be denatured and purified in vitro to obtain functional proteins with biological activity. At present, there are still problems such as high cost, low yield, and technical complexity in protein refolding technology [3], which greatly limits the application of inclusion body expression in actual production.

The discovery of active aggregates has changed the traditional view of inclusion bodies [4-6]. Studies have shown that protein aggregates with partial activity can be obtained by adjusting expression conditions or fused to express structural units that can cause aggregation. This insoluble, biologically active protein aggregate inherits the advantages of inclusion body expression, while avoiding complex denaturation operations.

The inventors found in previous studies that some amphiphilic self-assembled short peptides could induce the fusion protein to form active protein aggregates in E. coli when expressed in fusion with the target protein [7-9]. Such a self-assembled amphiphilic short peptide has simple secondary structure, high specific activity of the formed aggregate, and is very convenient to prepare.

The invention transforms the existing amphiphilic self-assembled short peptides, so that the distribution of active aggregates in cells can be changed, the growth state of host cells can be improved, and the yield of target proteins can be increased simultaneously. Such improvements expand the application of amphiphilic self-assembled short peptides in protein production and purification.

Summary of the invention

In a first aspect, the present invention provides a polypeptide comprising an amino acid sequence derived from SEQ ID NO: 1 which, compared to SEQ ID NO: 1, contains a polypeptide selected from the group consisting of K4E/E1K, K4E/E8K, K9E One or more double mutations of /E12K, K15E/E12K and K13E/E16K, wherein when the polypeptide is expressed in a host cell as a fusion protein with a protein of interest, the fusion protein can be formed in the host cell active aggregates.

In some embodiments, the polypeptide of the present invention comprises a polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 , the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

In some embodiments, when the polypeptide is expressed in a host cell as a fusion protein formed with a protein of interest, the protein yield of the fusion protein is comparable to that of a fusion protein formed by the polypeptide of SEQ ID NO:1 and the protein of interest protein production was increased compared to that of

In a second aspect, the present invention provides an isolated polynucleotide encoding the above-mentioned polypeptide of the present invention.

In the third aspect, the present invention provides a fusion protein comprising the protein of interest and the above-mentioned polypeptide of the present invention. In some embodiments, the protein of interest and the polypeptide are linked by a linker. In some embodiments, the linker comprises the sequence of SEQ ID NO:40. In other embodiments, the linker comprises a cleavage site. The cleavage site may be selected from a chemical cleavage site, an enzymatic cleavage site and a self-cleavage site, preferably a self-cleavage site. In some embodiments, the self-cleavage site is an intein, such as the intein whose sequence is set forth in SEQ ID NO:41.

In a fourth aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a fusion protein of the invention.

In a fifth aspect, the present invention provides an expression construct comprising the above-mentioned polynucleotide of the present invention.

In the sixth aspect, the present invention provides a host cell comprising the above-mentioned polynucleotide of the present invention or transformed with the above-mentioned expression construct of the present invention, wherein the host cell is capable of expressing the fusion protein.

In a seventh aspect, the present invention provides a method for producing and purifying a protein of interest, the method comprising the steps of:

(a) cultivating the above-mentioned host cell of the present invention, thereby expressing the fusion protein;

(b) lysing the host cells, then removing the soluble portion of the cell lysate and recovering the insoluble portion.

In some embodiments of the method for producing and purifying a target protein of the present invention, if the target protein in the fusion protein and the polypeptide of the present invention are connected through a linker containing a cleavage site, the method may also include the following steps:

(c) releasing soluble protein of interest from the insoluble fraction obtained in step (b) by cleaving said cleavage site;

(d) removing the insoluble portion in step (c), and recovering the soluble portion containing the target protein.

Description of drawings

Figure 1. Schematic diagram showing the structure of amphipathic α-helical self-assembled peptide and its interaction with phospholipids. A: The spiral wheel diagram of 18A; B: The spiral wheel diagram of 18Arev; C: Schematic diagram of the "Snorkel" model structure.

Fig. 2 shows amino acid pairs of a stable helix formed by lysine (K) at the 4th position from the N-terminal of 18A and glutamic acid (E) at the 1st position.

Figure 3. Shows the map of expression vectors for fusion proteins based on 18A variants. A: without cleavage site; B: with self-cleavage site.

Figure 4. Shows expression analysis of fusion proteins comprising 18A or 18A variants and LipA. A: Cell growth status; B: LipA enzyme activity assay results.

Figure 5. Results showing the distribution of GFP fusion protein aggregates comprising 18A or 18A variants in E. coli.

Figure 6. Shows a comparison of the intracellular distribution of fusion proteins of 18Av3, 18Av4, 18Av6 and 18Av8 with GFP and LipA.

Figure 7. Shows the results of the use of 18Arev and 18Av8 for the production and purification of LipA.

Figure 8. Shows the results of the use of 18Arev and 18Av8 for the production and purification of AMA.

Detailed description of the invention

The inventor's previous research found that the amphipathic α-helical self-assembling peptide 18A whose sequence is shown in SEQ ID NO:1 can mediate the fusion protein to form an insoluble protein in the cell after forming a fusion protein with the target protein and expressing it in the host cell. active aggregates. Active aggregates refer to the fact that the target protein in the aggregates can still maintain all or part of its biological activity despite being insoluble. "Amphipathic α-helical self-assembling peptide 18A" and "self-assembling peptide 18A" and "18A" are used interchangeably herein and have the same meaning.

The amphipathic α-helical self-assembling peptide 18A (as shown in Figure 1A) has the following characteristics: 1) Compared with ordinary α-helical peptides, it has a unique arrangement of hydrophilic and hydrophobic amino acids, so that one side of the formed α-helical structure is mainly hydrophilic Water-based amino acids, while on the other side are mostly hydrophobic amino acids. 2) According to the "Snorkel" model [10] (as shown in Figure 1C), the amphipathic α-helical self-assembling peptide 18A can bind to phospholipids or oils. This binding is mainly through hydrophobic interactions and electrostatic interactions between the positively charged lysine on 18A and the negatively charged phospholipid molecules.

The present inventors found that there are 5 pairs of amino acid pairs formed by positively charged amino acids and negatively charged amino acids adjacent to each other in space on the hydrophilic side of the helix of 18A: the 4th lysine from the N-terminus ( K) and glutamic acid (E) at the 1st position; lysine (K) at the 4th position from the N-terminal and glutamic acid (E) at the 8th position; lysine at the 9th position from the N-terminal acid (K) and glutamic acid (E) at position 12; lysine (K) at position 15 from the N-terminus and glutamic acid (E) at position 12; from the N-terminus at position 13 Lysine (K) and glutamic acid (E) at position 16.

Herein, the term "amino acid pair" refers to a pair of amino acids with complementary positive and negative charges and adjacent spatial positions in the α-helix. Herein, spatially adjacent means that two amino acid residues constituting an amino acid pair are spatially located in adjacent coils, and the interval between these two amino acid residues in the primary sequence is no more than 5 amino acid residues. FIG. 2 exemplarily shows an amino acid pair formed by lysine (K) at the 4th position from the N-terminal of 18A and glutamic acid (E) at the 1st position. Without being bound by any theory, it is speculated that a salt bridge can form between two residues in an amino acid pair, further stabilizing the alpha helical structure.

In this study, the inventors surprisingly found that the variant polypeptides obtained by mutating the 5 amino acid pairs of the amphipathic α-helical self-assembling peptide 18A separately or in combination can form fusion proteins with the target protein and express in host cells Finally, the fusion protein can also be induced to form active aggregates in cells, and at the same time, the spatial distribution of the formed active aggregates in cells is changed, which improves the growth state of host cells and increases the production of fusion proteins.

Accordingly, the present invention provides a polypeptide comprising an amino acid sequence derived from SEQ ID NO: 1 (18A), which, compared to SEQ ID NO: 1, contains a polypeptide selected from the group consisting of K4E/E1K, K4E/E8K, K9E One or more double mutations of /E12K, K15E/E12K and K13E/E16K. Such polypeptides are also referred to herein as "18A variants". When the 18A variant of the present invention is expressed in a host cell as a fusion protein with a target protein, the fusion protein can form active aggregates in the host cell.

The invention also provides an isolated polynucleotide comprising a nucleotide sequence encoding a 18A variant of the invention.

In this paper, the double mutation K4E/E1K means that the lysine (K) at the 4th position from the N-terminal of SEQ ID NO: 1 is mutated to glutamic acid (E), and the 1st position from the N-terminal of SEQ ID NO: 1 The glutamic acid (E) at the position is mutated to lysine (K); K4E/E8K means that the lysine (K) at the 4th position of SEQ ID NO:1 from the N-terminus is mutated to glutamic acid (E), and SEQ ID NO:1 The glutamic acid (E) at position 8 from the N-terminal of ID NO:1 is mutated to lysine (K); K9E/E12K represents the lysine (K) at position 9 from the N-terminal of SEQ ID NO:1 Mutation to glutamic acid (E), and the glutamic acid (E) at the 12th position from the N-terminal of SEQ ID NO:1 is mutated to lysine (K); K15E/E12K means that SEQ ID NO:1 is from the N-terminal The 15th lysine (K) is mutated to glutamic acid (E), and the 12th glutamic acid (E) from the N-terminal of SEQ ID NO: 1 is mutated to lysine (K); K13E/ E16K means that the lysine (K) at the 13th position from the N-terminal of SEQ ID NO: 1 is mutated to glutamic acid (E) and the glutamic acid (E) at the 16th position from the N-terminal of SEQ ID NO: 1 is mutated For lysine (K).

In some examples, the mutations contained in the 18A variant provided by the present invention, as well as the amino acid sequence and nucleotide sequence are shown in Table 1.

Table 1 Exemplary 18A variants

The mutations in the 18A variants of the present invention change the positively charged lysine on 18A to a negatively charged glutamic acid and simultaneously change the glutamic acid that forms a salt bridge with the lysine to a positively charged lysine , to maintain the stability of the salt bridge. Without being bound by any theory, charge inversion can produce effective repulsion, reducing or even eliminating the interaction between the 18A variant and the phospholipid molecule. Therefore, compared with 18A, the 18A variant of the present invention is capable of altering the distribution of its fusion protein aggregates in the host cell.

As shown in the following examples, generally, the more the number of lysine-glutamate pairs changed, the weaker the interaction between the 18A variant and the cell membrane phospholipid molecules, and the more likely the fusion protein aggregates are distributed in the cytoplasm. For example, when one amino acid pair is changed, 4/5 of the 18A variant fusion protein is distributed on the membrane; when two amino acid pairs are changed at the same time, 3/6 of the 18A variant fusion protein is distributed in the cytoplasm; When three or more amino acid pairs were changed, 5/8 of the 18A variant fusion protein was distributed in the cytoplasm. In addition, the stability of the α-helix itself also has a certain influence on the distribution of the fusion protein in the host cell. For example, it contains the mutation K4E/E1K (the 1st position of the N-terminal is a positively charged lysine), which is not conducive to the stability of the entire helix (the effect of dipoles), such 18A variants (such as 18Av6, 18Av7) fusion protein aggregates Tend to be distributed around the cell membrane; as another example, for the variant 18Av8, two pairs of salt bridges on its helix were destroyed (K4E/E1K, K15E/E12K), compared to 18Av9 and 18Av11 (only one pair of salt bridges was destroyed), The stability of 18Av8 is low, and the fusion protein aggregates containing this variant exhibit a characteristic distribution around the cell membrane.

The inventors have also surprisingly found that compared to 18A, the 18A variants of the present invention can improve the growth status of host cells expressing their fusion proteins. "Improving the growth state of host cells" means that the amount of host cells obtained after the same initial amount of host cells is cultured under the same culture conditions for the same time period increases.

Without being bound by any theory, the altered growth state of the host cells may be due to the altered intracellular distribution of fusion protein aggregates. As shown in the following examples, in general, for the 18A variant that can induce the fusion protein to distribute around the cell membrane, the growth state of the host cell expressing the corresponding fusion protein aggregate is worse than that of the host cell expressing the 18A fusion protein aggregate; For the 18A variant that can induce the fusion protein to distribute in the cytoplasm, the growth state of the host cells expressing the corresponding fusion protein aggregates is better than that of the host cells expressing the 18A fusion protein aggregates. However, there are a few exceptions, 18Av4, 18Av6, 18Av8, 18Av13, the growth state of the host cells containing the fusion protein aggregates of these four variants is better than that of the host cells containing the fusion protein aggregates of 18A, but the fusion protein induced by the four variants Aggregates are distributed wholly or partially around the cell membrane.

The growth status of the host cell expressing the fusion protein of the 18A variant is improved compared to the host cell expressing the 18A fusion protein. According to the comparison of Figures 4A and 4B, among the 12 18A variant fusion proteins that can improve the growth state of host cells, the total enzyme activity of 11 fusion proteins is higher than that of the 18A fusion protein; Among the 18A variant fusion proteins that were worse than 18A host cells, the total enzyme activity of 6 fusion proteins was lower than that of 18A fusion protein. This means that with the improvement of the cell growth state, the total enzyme activity of the fusion protein increases. According to the inventor's previous data [7], the fusion of LipA and 18A does not significantly change the enzyme activity of LipA itself, so the increase of the total enzyme activity of the fusion protein means the increase of the total yield of the fusion protein. That is to say, the growth state of the host cell is improved, which is beneficial to increase the total production of the fusion protein. Correspondingly, the yield of the protein of interest can also be increased (see, eg, Examples 6 and 7).

Another aspect of the present invention provides a fusion protein comprising a protein of interest and a 18A variant, wherein the 18A variant comprises an amino acid sequence derived from SEQ ID NO: 1, which is identical to SEQ ID NO: 1 The comparison contains one or more double mutations selected from K4E/E1K, K4E/E8K, K9E/E12K, K15E/E12K and K13E/E16K. The fusion protein can form active aggregates after being expressed in host cells, and its protein yield is higher than that of the fusion protein formed by the target protein and 18A.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein. Herein, "target protein" refers to any polypeptide or protein that can be expressed in fusion with the 18A variant of the present invention and form active aggregates. The protein of interest can come from any source, including microbial-derived polypeptides, mammalian-derived polypeptides, artificial proteins (such as chimeric or mutated proteins), and the like.

In some embodiments, the protein of interest in the fusion protein of the present invention is connected to the 18A variant through a linker. As used herein, "linker" refers to a polypeptide with a certain length consisting of amino acids with low hydrophobicity and low charge effect. When it is used in a fusion protein, it can fully unfold the connected parts and fully fold into respective native conformations. Such linkers commonly used in the art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); rigid PT-type linkers rich in proline (P) and threonine (T). Since PT-type linkers generally have better protease resistance than GS-type linkers, PT-type linkers are preferably used in the present invention. In some embodiments, the linker used in the present invention comprises the sequence PTPPTTPTPPTTPTPT (SEQ ID NO: 40).

In some embodiments, a linker in a fusion protein of the invention may also comprise a cleavage site. By cleaving the cleavage site, the protein of interest can be separated from the aggregate. Suitable cleavage sites include cleavage sites that can be chemically cleaved, enzymatically cleaved, or self-cleaved, or any other cleavage site known to those skilled in the art. Preferred cleavage sites in the present invention are self-cleavable, for example, comprise the amino acid sequence of a self-cleavable intein. This is because the intein-based cleavage method does not require external enzymes or use of harmful substances such as hydrogen bromide used in chemical methods, but simply induces cleavage by changing the buffer environment in which the aggregates are placed [11,12 ]. A variety of self-cleaving inteins are known in the art, for example, a series of inteins with different self-cleaving properties from NEB Company. In a specific embodiment, the intein is MxeGyrA whose sequence is shown in SEQ ID NO: 41, and the intein can be induced in the buffer system by adding an appropriate amount of dithiothreitol (DTT). Self-cleavage of its carboxy terminus.

In another aspect, the present invention also provides an isolated polynucleotide comprising a nucleotide sequence encoding the fusion protein of the present invention. The invention also provides expression constructs comprising such polynucleotides.

As used herein, "polynucleotide" refers to a macromolecule composed of multiple nucleotides, including ribonucleotides and deoxyribonucleosides, linked by 3'-5'-phosphodiester bonds acid. The sequence of the polynucleotide of the present invention can be codon-optimized for different host cells (such as Escherichia coli), so as to improve the expression of the fusion protein. Methods for performing codon optimization are known in the art.

In the expression construct of the present invention, the sequence of the polynucleotide encoding the fusion protein of the present invention is operably linked to expression control sequences for the desired transcription and ultimately production of the fusion protein in a host cell. Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosomal interaction sites such as ribosomal binding sites, polyadenylation sites, transcriptional splicing sequences, transcriptional termination sequences, sequences that stabilize mRNA, and the like.

Vectors used in the expression constructs of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; and also include vectors that are capable of integrating into and replicating with host cell DNA. Many vectors suitable for the present invention are commercially available. In a specific embodiment, the expression construct of the present invention is derived from pET30a(+) from Novagen.

In another aspect, the present invention provides a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a fusion protein of the present invention or transformed with an expression construct comprising said polynucleotide, wherein said host cell capable of expressing the fusion protein of the present invention.

The host cells used to express the fusion protein of the present invention include prokaryotes, yeast and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, Salmonella, and the genera Pseudomonas and Streptomyces. In a preferred embodiment, the host cell is an Escherichia cell, preferably E. coli. In a specific embodiment of the present invention, the host cell used is Escherichia coli BL21(DE3) strain cell (Novagen).

The recombinant expression constructs of the present invention can be introduced into host cells by one of many well-known techniques including, but not limited to: heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, lipid Infection-mediated transfection, calcium phosphate precipitation, protoplast fusion, particle bombardment, viral transformation and similar techniques.

In another aspect, the present invention provides a method for producing and purifying a protein of interest, said method comprising the steps of:

(a) cultivating the above-mentioned host cell of the present invention, thereby expressing a fusion protein comprising the 18A variant of the present invention and the protein of interest;

(b) lysing the host cells, then removing the soluble portion of the cell lysate and recovering the insoluble portion.

In other embodiments, if the protein of interest in the fusion protein and the 18A variant are connected through a linker containing a cleavage site, the method may further include the following steps:

(c) releasing soluble protein of interest from the insoluble fraction obtained in step (b) by cleaving said cleavage site;

(d) removing the insoluble portion in step (c), and reclaiming the soluble portion containing the protein of interest.

In the method for producing and purifying the target protein of the present invention, after the 18A variant capable of inducing the formation of active aggregates and the target protein is expressed in a host cell as a fusion protein, the expressed fusion protein forms an insoluble aggregate. The formation of aggregates can avoid the degradation of fusion proteins by intracellular proteases. After cell lysis, insoluble aggregates can be collected from the cell lysate simply by centrifugation or filtration to remove soluble impurities and achieve preliminary purification of the fusion protein. Thereafter, by cleavage of the cleavage site located in the linker between the 18A variant of the invention and the protein of interest, the soluble fraction containing the protein of interest is released from the insoluble fraction (precipitate) and distributed in the supernatant, Once again, the insoluble impurities can be removed simply by centrifugation or filtration, and the soluble target protein can be harvested. The production of protein by such a method based on the 18A variant that can cause aggregation can simplify the separation and purification steps, avoid the use of expensive purification columns, and significantly reduce production costs. The present inventors found that compared with 18A, the yield of the target protein can be significantly improved by using the 18A variant of the present invention.

Example

The present invention will be further illustrated by means of examples below, but the present invention is not limited to the scope of the described examples. The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in, for example, "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). All primers used were synthesized by Invitrogen.

Example 1: Construction of a fusion protein expression vector using Bacillus subtilis lipase A (LipA) and green fluorescent protein (GFP) as target proteins

1.1 Amplification of polynucleotide fragments of 18A variants (19 species)

Firstly, the nucleotide sequence of the PT-type linker and the 18A variant was designed with the online tool DNAworks. The oligonucleotide primers shown in Table 2 were designed by DNAWorks [13] and synthesized, and then the complete sequence encoding the 18A variant (Hind III-linker-18A variant-Xho I) was obtained by overlapping PCR (overlapping PCR) method. polynucleotide sequence.

Table 2 is used to amplify the primer list of 18A variant

^a The underlined parts of the primers are the recognition sites of restriction endonucleases Hind III and Xho I, respectively.

Taking one of the variants, 18Av1, as an example, the specific method for amplifying the polynucleotide fragment encoding the 18A variant will be described below.

DNAWorks output is used to synthesize 4 overlapping oligonucleotide fragments of the PT linker 18Av1 gene (including a set of primers for the PT linker and a set of primers for 18Av1, and the sequences are shown in Table 2). These oligonucleotide fragments were synthesized by Invitrogen.

The synthesized oligonucleotide fragments were dissolved with 10 mM Tris-HCl, pH 8.0 buffer solution, the PCR reaction solution was prepared according to Table 3, and then the PCR reaction was carried out according to the reaction procedure in Table 4.

Table 3: PCR reaction system

Reactant volume Oligonucleotide Fragment Mixture The final concentration of each fragment is 625nM 5× fast pfu buffer 10μL Fast pfu 1μL dNTPs 5μL double distilled water 32μL total 50μL

Table 4: PCR reaction program

step temperature time 1 95°C 5min

2 95°C 20s 3 59°C 20s 4 72°C 15s 5 return to step 2 24 times 6 72°C 5min 7 4°C long time 8 End

Using the PCR product obtained above as a template, use the following forward primer and reverse primer to perform PCR amplification according to the conventional method to obtain the PT linker-18Av1 nucleotide fragment: upstream primer 5'-ATGAA AAGCTT CCGACCCACCGACCAC-3' (SEQ ID NO:42, the underlined base is the restriction endonuclease Hind III recognition site), and the downstream primer 5'- CTCGAG TCAGAACAGTTTCTTTCAGTTTCTCAGGACCTTTTCGTAGAACGCTTC-3' (SEQ ID NO:43, the underlined base is the restriction endonuclease Xho I recognition site). Prepare the PCR reaction solution according to the following Table 5, and then amplify the PT linker-18Av1 gene according to the reaction program in the following Table 6.

Table 5: PCR reaction system for amplification

Reactant Amount added template (assembly product) 2μL 5× fast pfu buffer 20 μL dNTPs 8μL Upstream primer (20μM) 4μL Downstream primer (20μM) 4μL Fast pfu 2μL double distilled water 60μL total 100μL

Table 6: Amplification PCR reaction program

step temperature time 1 95°C 5min 2 95°C 20s 3 59°C 20s 4 72°C 15s 5 return to step 2 29 times 6 72°C 5min 7 4°C long time 8 End

After the reaction was completed, the PCR amplification product was detected by 1% agarose gel electrophoresis, and as a result, the correct band was amplified by PCR, which was consistent with the expectation.

1.2 Construction of fusion protein expression vectors of LipA/GFP and 18A/18A variants, and non-fusion LipA/GFP protein expression vectors

The construction process of the expression vector pET-30a(+)-LipA-18A/18A variant and pET-30a(+)-GFP-18A/18A variant used in the examples of the present application: will be overlapped by Example 1.1 The PCR product was double-digested with restriction endonucleases Hind III and Xho I, respectively, and the plasmids pET-30a(+)-LipA-ELK16 and pET-30a(+)-GFP-ELK16 ( These two vectors are constructed but not disclosed by the inventors for the fusion expression of LipA, GFP and another self-assembling short peptide ELK16 tag [8] that can cause aggregation. The full-length sequence of the plasmid can be found in SEQ IDNO:82 and SEQ ID NO:83) were ligated, and the ELK16 tag was replaced with the 18A/18A variant to obtain a fusion expression vector.

Taking pET-30a(+)-LipA-18Av1 and pET-30a(+)-GFP-18Av1 as examples, the specific construction methods are described below:

First construct the pET-30a(+)-LipA-18Av1 vector, the vector structure is shown in Figure 3A, wherein the "Targetprotein" sequence is the sequence of Bacillus subtilis lipase A (LipA) (SEQ ID NO: 84). The overlapping PCR product Hind III-linker-18Av1-Xho I obtained in the above steps was double-digested with restriction endonucleases Hind III and Xho I, and the plasmid pET-30a(+)-LipA was double-digested with the same enzymes -ELK16 (SEQ ID NO:82) was ligated, and the ligated product was transformed into Escherichia coli BL21(DE3) (Novagen) competent cells, and the transformed cells were screened on LB plates supplemented with 50 μg/mL kanamycin For positive clones, the plasmids were extracted and sequenced. The sequencing results showed that the sequence of the cloned pET-30a(+)-LipA-18Av1 was correct.

Similarly, for the pET-30a(+)-GFP-18Av1 vector, the vector structure is shown in Figure 3A, wherein the "Targetprotein" sequence is the sequence of green fluorescent protein (GFP) (SEQ ID NO: 85). The overlapping PCR product Hind III-linker-18Av1-Xho I obtained in the above steps was double-digested with restriction endonucleases Hind III and Xho I, and then combined with the plasmid pET-30a(+)-GFP double-digested with the same enzymes -ELK16 (SEQ ID NO:83) was ligated, and the ligated product was transformed into Escherichia coli BL21(DE3) (Novagen) competent cells, and the transformed cells were screened on LB plates supplemented with 50 μg/mL kanamycin For positive clones, the plasmids were extracted and sequenced. The sequencing results showed that the sequence of the cloned pET-30a(+)-GFP-18Av1 was correct.

In addition, the expression vectors pET-30a(+)-LipA-18A and pET-30a(+)-GFP-18A used in the examples of this application, and the expression vector pET-30a(+)-LipA-native of the control protein and pET-30a(+)-GFP-native are plasmids that the inventor has constructed but not disclosed, and their full-length sequences are respectively SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO :89. Those skilled in the art can easily prepare these plasmids.

Example 2: Expression, cell growth status and enzyme activity assay of a fusion protein with Bacillus subtilis lipase A (LipA) as the target protein

2.1 Induced expression of fusion protein

The strain constructed in Example 1 (containing plasmids pET-30a(+)-LipA-native, pET-30a(+)-GFP-18A and pET-30a(+)-GFP-18A variants) was inoculated into the 50 μg/mL kanamycin in LB liquid medium, and cultured in a shaker at 37°C to the logarithmic phase (OD ₆₀₀ =0.4-0.6), added 0.2mM IPTG, induced at 30°C for 6 hours, and harvested the cells , and measure the bacterial concentration OD ₆₀₀ (hereafter, the amount of cells whose OD ₆₀₀ is 1 in 1 mL is referred to as 1OD).

2.2 Cell growth state

The growth state of cells expressing the LipA-18A variant fusion protein is shown in Table 7 and Figure 4A below.

Table 7 expresses the OD ₆₀₀ value of LipA-18A and LipA-18A variant fusion protein cells

18A variant LipA-18A LipA-18Arev LipA-18Av1 LipA-18Av2 LipA-18Av3 OD 1.57 2.82 0.98 1.05 1.49 18A variant LipA-18Av4 LipA-18Av5 LipA-18Av6 LipA-18Av7 LipA-18Av8 OD 2.18 1.1 1.81 1.2 2.39 18A variant LipA-18Av9 LipA-18Av10 LipA-18Av11 LipA-18Av12 LipA-18Av13 OD 2.1 1.64 2.34 0.67 1.88 18A variant LipA-18Av14 LipA-18Av15 LipA-18Av16 LipA-18Av17 LipA-18Av18 OD 2.77 2.85 2.58 2.83 0.88

Cells expressing 12 of the LipA-18A variant fusion proteins grew better than cells expressing LipA-18A fusion proteins (higher OD ₆₀₀ values): LipA-18Av9, LipA-18Av10, LipA-18Av11, LipA-18Av14, LipA-18Av15, LipA-18Av16, LipA-18Av17, LipA-18Arev and LipA-18Av4, LipA-18Av6, LipA-18Av8, LipA-18Av13. The growth status of cells expressing seven of the LipA-18A variant fusion proteins was comparable to or worse than that of LipA-18A: LipA-18Av1, LipA-18Av2, LipA-18Av3, LipA-18Av5, LipA-18Av7, LipA -18Av12, LipA-18Av18.

2.3 Enzyme activity assay

Harvest a unit volume (1 liter of LB liquid medium) and resuspend the above induced cells with lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH 7.2). Cells were disrupted by ultrasonication on ice (disruption conditions: power 200W, ultrasonic time 3s, interval time 3s, ultrasonic frequency 99 times). After sonication is complete, carefully separate the supernatant and pellet of the lysate by centrifugation. In order to remove the mixed soluble components in the pellet as much as possible, the obtained pellet was washed twice with an equal volume of lysis buffer. The supernatant and pellet resuspension were directly used for the corresponding enzyme activity assays. Wherein, the quantitative assay method of lipase activity is as follows:

The activity of LipA on p-nitrophenyl palmitate (pNPP) was determined. For the pNPP measurement method, please refer to the literature (Winkler, U.K., M.Stuckmann, Glycogen, Hyaluronate, and Some Other Polysaccharides Greatly Enhance the Formation of Exolipase by Serratiamarcescens, JOURNAL OF BACTERIOLOGY, 1979, 138(3):663-670). The activity is defined as: under the assay conditions, the amount of enzyme required to hydrolyze the above substrate to produce 1 nmol of p-nitrophenol (p-nitrophenol) or fatty acid (fattyacid) within 1 minute is defined as 1 activity unit.

The enzyme activity data of the fusion protein are shown in Figure 4B. As with the LipA-18A fusion protein, for most of the variant fusion proteins, 70-97% of the enzyme activity was distributed in the pellet (except for LipA-18Av11 and LipA-18Av16, the enzyme activity of these two fusion proteins in the pellet activity accounted for 65% and 54% of the total enzyme activity respectively). Among these variant fusion proteins, 12 fusion proteins have higher total enzyme activity than LipA-18A, they are (from high to low activity): LipA-18Av8, LipA-18Av15, LipA-18Av17, LipA-18Arev, LipA-18Av6, LipA-18Av14, LipA-18Av9, LipA-18Av11, LipA-18Av16, LipA-18Av4, LipA-18Av3, LipA-18Av10. Among them, the total enzyme activity of LipA-18Av8 is about 2.9 times of that of LipA-18A.

The results show that all 19 18A variants can cause the aggregation of LipA protein, and the aggregates formed can retain the activity of LipA protein to varying degrees, among which 12 variants are beneficial to the improvement of the total enzyme activity in bacteria expressing LipA fusion protein .

Combining cell growth state data and total enzyme activity data (Figure 4A and B), among the 12 18A variant fusion proteins that can improve the growth state of host cells, 11 fusion proteins have higher total enzyme activity than 18A fusion proteins; Among the 7 18A variant fusion proteins that caused host cells to grow worse than 18A host cells, the total enzyme activity of 6 fusion proteins was lower than that of 18A fusion proteins. This means that with the improvement of the cell growth state, the total enzyme activity of the fusion protein increases.

Example 3: Expression of fusion protein with green fluorescent protein (GFP) as the target protein and its intracellular distribution

The strain constructed in Example 1 was inoculated into LB liquid medium containing 50 μg/mL kanamycin, and cultured in a shaker at 37°C to the logarithmic phase (OD ₆₀₀ =0.4-0.6), adding 0.2mM IPTG, induced at 23°C for 22 hours, and cells were harvested.

The harvested cells were treated with 4% paraformaldehyde at 4°C for 1 h. Fluorescent confocal microscopy of GFP cells was performed on a Zeiss710 inverted confocal microscope (Zeiss LSM710confocal microscope) with an excitation wavelength of 488nm.

The intracellular distribution of active enzyme aggregates induced by 18A and 18A variants is shown in Figure 5. It can be clearly seen from the fluorescent photos that the fluorescence of cells expressing GFP-18A fusion protein is mainly distributed on the inner side of the cell membrane. It shows that the GFP-18A fusion protein aggregates are distributed on the inner side of the cell membrane. For cells expressing fusion proteins containing 18A variants, the distribution of 8 18A variant fusion protein aggregates is similar to GFP-18A (distributed inside the cell membrane), which are GFP-18Av1, GFP-18Av2, GFP-18Av4 , GFP-18Av5, GFP-18Av6, GFP-18Av7, GFP-18Av12, GFP-18Av13. There are also 8 kinds of 18A variant fusion protein aggregates whose distribution is completely different from GFP-18A (distributed in the cytoplasm of the cell, can be located at one end or both ends of the cell, and the shape is similar to a typical inclusion body), they are GFP-18Av9, GFP-18Av10, GFP-18Av11, GFP-18Av14, GFP-18Av15, GFP-18Av16, GFP-18Av17, and GFP-18Arev. There are three 18A variant fusion protein aggregates showing a partial distribution inside the cell membrane and a partial distribution in the cytoplasm, which are GFP-18Av3, GFP-18Av8, and GFP-18Av18.

The above results indicated that the above-mentioned 18A variant could induce the green fluorescent protein GFP to form biologically active protein aggregates in cells, and could change the distribution of GFP in Escherichia coli.

Combining the results of the intracellular distribution of the GFP-18A variant fusion protein and the growth status of cells expressing the LipA-18A variant fusion protein, it can be known that: for the 18A variants that can induce the fusion protein to distribute around the cell membrane (LipA-18Av1, LipA- 18Av2, LipA-18Av3, LipA-18Av5, LipA-18Av7, LipA-18Av12, LipA-18Av18), the growth status of the host cells expressing the corresponding LipA fusion protein was worse than that of the host cells expressing LipA-18A; while for the inducible fusion protein The 18A variants (LipA-18Av9, LipA-18Av10, LipA-18Av11, LipA-18Av14, LipA-18Av15, LipA-18Av16, LipA-18Av17, LipA-18Arev) whose protein is distributed in the cytoplasm express the corresponding LipA fusion protein The growth status of the host cells is better than that of the host cells expressing LipA-18A. With only a few exceptions, LipA-18Av4, LipA-18Av6, LipA-18Av8, LipA-18Av13, host cells containing these four fusion proteins grew better than those containing LipA-18A, but these four variants induced GFP fusion proteins All or part of the distribution around the cell membrane. The results showed that when the expressed active aggregates were distributed in the cytoplasm, it was beneficial to cell growth.

Example 4: Comparing the intracellular distribution of LipA-18A variant fusion protein and GFP-18A variant fusion protein

In order to confirm the consistency of the intracellular distribution of the LipA-18A variant fusion protein with the corresponding GFP fusion protein distribution in the cell, the inventors selected 4 species expressing LipA-18Av3, LipA-18Av4, LipA-18Av6 and LipA -18Av8 fusion protein Escherichia coli was subjected to ultra-thin cell sections, the distribution of LipA fusion protein in the cell was observed with a transmission electron microscope, and compared with the distribution results of the corresponding GFP fusion protein in the cell.

The experimental method is as follows:

The corresponding bacterial strain constructed in Example 1 (containing plasmids pET-30a (+)-LipA-18Av3, pET-30a (+)-LipA-18Av4, pET-30a (+)-LipA-18Av6, pET-30a (+)-LipA-18Av8) were inoculated into LB liquid medium containing 50g/mL kanamycin, and cultured in a shaker at 37°C to logarithmic phase (OD ₆₀₀ =0.4-0.6), adding 0.2mM IPTG , induced at 30°C for 6 hours, and the cells were harvested.

Cells were fixed by adding 2.5% glutaraldehyde solution and 2% osmium tetraoxide solution in turn. The fixed cells were dehydrated with a series of ethanol concentrations (30%, 50%, 70%, 90%, 100%), and then embedded with epoxy resin. Ultrathin cell sections were obtained using an ultramicrotome (Lecia EM UC6), and then stained with uranyl acetate solution and lead citrate solution for a certain period of time, observed in a Hitachi H-7650B transmission electron microscope, electron acceleration The voltage is 80kV.

The experimental results are as follows:

For 18Av3, the distribution of its fusion protein is shown in Figure 6A. TEM results (left) showed that part of the LipA-18Av3 fusion protein formed aggregates inside the cell membrane (arrows indicate fusion protein aggregates), and part of LipA-18Av3 fusion proteins formed aggregates in the cytoplasm (arrows indicated fusion protein aggregates) . This result is consistent with the intracellular distribution of GFP fusion protein (right).

For 18Av4, the distribution of its fusion protein is shown in Figure 6B. TEM results (left) showed that the LipA-18Av4 fusion protein formed a layer of aggregates inside the cell membrane (arrows indicate fusion protein aggregates). This result is consistent with the intracellular distribution of GFP fusion protein (right).

The distribution of the 18Av6 fusion protein was similar to that of the 18Av4 fusion protein, as shown in Figure 6C. The results of TEM (left) showed that the LipA-18Av6 fusion protein also formed a layer of aggregates inside the cell membrane (arrows indicate fusion protein aggregates). This result is consistent with the intracellular distribution of GFP fusion protein (right).

For 18Av8, the distribution of its fusion protein is shown in Figure 6D. TEM results (left) showed that part of the LipA-18Av8 fusion protein formed aggregates inside the cell membrane (arrows indicate fusion protein aggregates), and part of LipA-18Av8 fusion proteins formed aggregates at one end of the cell (arrows indicated fusion protein aggregation body). This result is consistent with the intracellular distribution of GFP fusion protein (right).

The above results indicate that the results obtained from the GFP fusion protein experiment can represent the distribution of fusion proteins of 18A variants and other proteins in cells.

Example 5: Construction of a fusion protein expression vector containing a cleavage site with Bacillus subtilis lipase A (LipA) and Aspergillus fumigatus Amadoriase II (AMA) as target proteins

Bacillus subtilis lipase A (LipA) and Aspergillus fumigatus Amadoriase II (AMA) were respectively selected as the target proteins, and the two 18A variants 18Arev and 18Av8 of the present invention were used to construct fusion proteins additionally containing the Mxe GyrA self-cleavage site, and The production and purification of the target protein is carried out by the method of the present invention.

The construction process of the expression vector pET-30a(+)-LipA-Mxe-18Arev/18Av8 and pET-30a(+)-AMA-Mxe-18Arev/18Av8 used in the examples of the present application: the overlapping PCR obtained from Example 1 The product was double digested with restriction endonucleases Hind III and Xho I, and the plasmid pET-30a(+)-LipA-Mxe-ELK16 and pET-30a(+)-AMA-Mxe- ELK16 (a plasmid constructed but not disclosed by the inventor, the full-length sequence of SEQ ID NO:90 and SEQ ID NO:91) was ligated to obtain the desired fusion expression vector (see Figure 3B for a schematic diagram). The ligation product was transformed into Escherichia coli BL21(DE3) (Novagen) competent cells, the transformed cells were spread on the LB plate supplemented with 50 μg/mL kanamycin to screen positive clones, the plasmid was extracted, sequenced, and sequenced The results showed that the cloned sequences of pET-30a(+)-LipA-Mxe-18Arev/18Av8 and pET-30a(+)-AMA-Mxe-18A rev/18Av8 were correct.

In addition, the expression vectors pET-30a(+)-LipA-Mxe-ELK16, pET-30a(+)-AMA-Mxe-ELK16, pET-30a(+)-LipA-ELK16, pET-30a(+)-LipA- Mxe-18A and pET-30a(+)-AMA-Mxe-18A are all plasmids constructed but not disclosed by the inventors, and their full-length sequences are respectively SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93. Those skilled in the art can easily prepare these plasmids.

Example 6: Expression and purification of fusion proteins LipA-Mxe-18Arev and LipA-Mxe-18Av8

Inoculate the strains containing expression vectors pET-30a(+)-LipA-Mxe-18Arev/18Av8 and pET-30a(+)-LipA-Mxe-ELK16/18A respectively into LB liquid culture containing 50 μg/mL kanamycin 0.2 mM IPTG was added to the base, induced at 30°C for 6 hours, and the cells were harvested. And measure the bacterial concentration OD ₆₀₀ (hereafter, the amount of cells whose OD ₆₀₀ is 1 in 1 mL is referred to as 1OD).

Resuspend the cells with lysis buffer B1 (dissolve 2.4g Tris, 29.22g NaCl, 0.37g Na2EDTA _· 2H2O in 800mL water, adjust the pH to _8.2 , add water to 1L) to 10OD/ mL, sonicated. Centrifuge at 4°C and 10,000 rpm for 10 min, and collect the supernatant and precipitate, respectively. After the pellet was washed twice with lysis buffer, it was fully resuspended in cleavage buffer (20mM Tris-HCl (pH8.0), 500mM NaCl, 40mM dithiothreitol, 1mM EDTA), and placed at 4°C overnight for 24h. The intein is fully self-cleaved. Afterwards, the suspension was centrifuged, and the resulting supernatant and pellet were subjected to SDS-PAGE detection together with the pellet before cutting (the pellet was resuspended with the same volume of lysis buffer as in the previous resuspension step). The result is shown in Figure 7. Swimming lane 1: cell lysate pellet before cleavage, clear enzyme aggregates expressed by ternary fusion protein can be detected; Swimming lane 2: supernatant separated after cleavage, clear target protein band can be detected; Swimming lane 3-5: protein quantification standard substance containing bovine serum albumin BSA, the loading amount is 6 μg, 3 μg, 1.5 μg and 0.75 μg in sequence.

According to the protein quantification standard, apply Bio-Rad's Quantity ONE gel quantitative analysis software to analyze the optical density of the target band, and calculate the aggregate yield formed by the fusion protein, after intein-mediated self-cleavage The results of the yield of the target protein released into the supernatant and the purity of the target polypeptide in the supernatant are shown in Table 8.

Table 8 The expression level of the LipA fusion protein aggregate and the yield of the target protein

^a . Aggregate yield and ^b . Intein-mediated target protein yield after self-cleavage (calculated as 2.66 mg wet cell weight per ml of E. coli cells in LB medium when the bacterial concentration OD ₆₀₀ is 2).

For LipA fusion protein (SDS-PAGE results are shown in Figure 7), four self-assembled short peptides: ELK16, 18A, 18Arev and 18Av8 can form a large amount of aggregates after fusion expression with LipA, and the expression level is 24.4-34.1μg/mg cells Wet weight; after intein-mediated self-cleavage, the target protein LipA can be released into the supernatant, and the yield is 8.3-15.1 μg/mg cell wet weight. Considering that the expression of different self-assembled short peptide fusion proteins has an impact on the OD value of cells (compared with 18A, 18Arev and 18Av8 are beneficial to the growth of cells), we calculated the yield of aggregates and target proteins per liter of bacterial liquid. For the three fusion proteins of ELK16, 18Arev and 18Av8, the expression level of aggregates was 96.7-98.1 mg/L bacterial fluid, which was significantly higher than the expression level of 18A fusion protein aggregates (33.3 mg/L bacterial fluid); The output of the target protein LipA that can be released into the supernatant after mediated self-cleavage is 30.6-54mg/L bacterial fluid, wherein the output of LipA released by the 18Av8 fusion protein is equivalent to that of 18Arev, and its output is 1.8 times that of the ELK16 fusion protein, which is 5.4 times that of 18A fusion protein.

Example 7: Expression and cleavage purification of fusion proteins AMA-Mxe-18Arev and AMA-Mxe-18Av8

Inoculate the strains containing expression vectors pET-30a(+)-AMA-Mxe-18Arev/18Av8 and pET-30a(+)-AMA-Mxe-ELK16/18A respectively into LB liquid culture containing 50 μg/mL kanamycin 0.2mM IPTG was added to the base, induced at 30°C for 6 hours, and the cells were harvested. And measure the bacterial concentration OD ₆₀₀ (hereinafter, the amount of cells whose OD ₆₀₀ is 1 in 1 mL is referred to as 1OD).

Resuspend the cells with lysis buffer B1 (dissolve 2.4g Tris, 29.22g NaCl, 0.37g Na2EDTA _· 2H2O in 800mL water, adjust the pH to _8.2 , add water to 1L) to 10OD/ mL, sonicated. Centrifuge at 4°C and 10,000 rpm for 10 min, and collect the supernatant and precipitate, respectively. After the pellet was washed twice with lysis buffer, it was fully resuspended in cleavage buffer (20mM Tris-HCl (pH8.0), 500mM NaCl, 40mM dithiothreitol, 1mM EDTA), and placed at 4°C overnight for 24h. The intein is fully self-cleaved. Afterwards, the suspension was centrifuged, and the resulting supernatant and pellet were subjected to SDS-PAGE detection together with the pellet before cutting (the pellet was resuspended with the same volume of lysis buffer as in the previous resuspension step). The result is shown in Figure 8. Swimming lane 1: cell lysate pellet before cleavage, and clear enzyme aggregates formed by expression of fusion protein triplet can be detected; Swimming lane 2: supernatant separated after cleavage, clear target protein band can be detected; 3-5: Protein quantitative standard containing bovine serum albumin BSA, the loading amount is 6 μg, 3 μg, 1.5 μg and 0.75 μg in sequence.

According to the protein quantification standard, apply Bio-Rad's Quantity ONE gel quantitative analysis software to analyze the optical density of the target band, and calculate the aggregate yield formed by the fusion protein, after intein-mediated self-cleavage The yield of the target protein released into the supernatant and the purity of the target polypeptide in the supernatant are shown in Table 9.

Table 9 The expression level of the LipA fusion protein aggregate and the yield of the target protein

^a protein aggregate yield and ^b intein-mediated self-cleavage target polypeptide yield (calculated as 2.66 mg cell wet weight per milliliter of E. coli cells in LB medium when the bacterial concentration OD ₆₀₀ is 2).

For the AMA fusion protein (see Figure 8 for SDS-PAGE results), four self-assembled short peptides: ELK16, 18A, 18Arev and 18Av8 can form a large amount of aggregates after fusion expression with AMA, and the expression level is 19.1-25.5μg/mg cells Wet weight; after intein-mediated self-cleavage, the target protein LipA can be released into the supernatant, and the yield is 4.0-7.9 μg/mg wet weight of cells. Considering that the expression of different self-assembled short peptide fusion proteins has a certain impact on the OD value of the cells (compared with 18A, 18Arev and 18Av8 are beneficial to the growth of cells), we calculated the yield of aggregates and target proteins per liter of bacteria solution . For the three fusion proteins of ELK16, 18Arev and 18Av8, the expression level of aggregates was 65.3-86.9 mg/L bacterial fluid, which was significantly higher than the expression level of 18A fusion protein aggregates (35.3 mg/L bacterial fluid); The yield of the target protein AMA that can be released into the supernatant after mediated self-cleavage is 8.2-24mg/L bacterial fluid, among which the yield of AMA released by the 18Av8 fusion protein is the highest, which is 2.9 times that of the ELK16 fusion protein, which is higher than that of the 18A fusion protein. 1.8 times of protein release.

references

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Claims (16)

1. a kind of polypeptide, it is by derived from SEQ ID NO:1 amino acid sequence composition, the amino acid sequence and SEQ ID NO:1 compares containing the double mutation of K4E/E1K, or containing K4E/E1K and selected from K4E/E8K, K9E/E12K, K15E/E12K and The double mutation of one or more of K13E/E16K, wherein when the polypeptide and the fusion protein of destination protein formation are in host cell During expression, the fusion protein can form active aggregation in the host cell.

2. the polypeptide of claim 1, it is by selected from SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO: 13、SEQ ID NO:14、SEQ ID NO:19 and SEQ ID NO:20 amino acid sequence composition.

3. the polypeptide of claim 1 or 2, wherein the table in host cell when the polypeptide and the fusion protein of destination protein formation Up to when, the protein yield of the fusion protein with by SEQ ID NO:The fusion protein that 1 polypeptide is formed with the destination protein Protein yield compared to being increased.

4. the polynucleotides of separation, it encodes any one of claim 1-3 polypeptide.

5. fusion protein, it includes any one of destination protein and claim 1-3 polypeptide.

6. the fusion protein of claim 5, wherein the destination protein is connected with the polypeptide by joint.

7. the fusion protein of claim 6, wherein the joint includes SEQ ID NO:40 sequence.

8. the fusion protein of claim 6, wherein the joint includes cleavage site.

9. the fusion protein of claim 8, wherein the cleavage site is selected from chemical cleavage site, enzyme process cleavage site and autotomyed Cut site.

10. the fusion protein of claim 9, wherein Self cleavage site are intein.

11. the fusion protein of claim 10, wherein the sequence of the intein is shown in SEQ ID NO:41.

12. the polynucleotides of separation, it includes the nucleotide sequence of any one of coding claim 5-11 fusion protein.

13. expression construct, it includes the polynucleotides of claim 12.

14. host cell, it is included the polynucleotides of claim 12 or converted with the expression construct of claim 13, wherein The host cell can express the fusion protein.

15. the method for producing and purifying destination protein, the described method comprises the following steps：

(a) host cell of claim 14 is cultivated, so as to express the fusion protein；With

(b) host cell is cracked, the soluble fraction of cell lysate is then removed, reclaims insoluble part.

16. the method for claim 15, wherein the destination protein and the polypeptide in the fusion protein are by containing cutting The joint for cutting site is connected, and methods described is further comprising the steps of：

(c) solvable destination protein is discharged from the insoluble part that step (b) obtains by cutting the cleavage site；

(d) the insoluble part in removal step (c), soluble fraction of the recovery containing the destination protein.