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WO1992019737A1 - Procede de preparation d'un polypeptide de facteur chimiotactique de monocyte et souche utilisee pour cette preparation - Google Patents

Procede de preparation d'un polypeptide de facteur chimiotactique de monocyte et souche utilisee pour cette preparation Download PDF

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Publication number
WO1992019737A1
WO1992019737A1 PCT/JP1992/000550 JP9200550W WO9219737A1 WO 1992019737 A1 WO1992019737 A1 WO 1992019737A1 JP 9200550 W JP9200550 W JP 9200550W WO 9219737 A1 WO9219737 A1 WO 9219737A1
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coli
strain
sequence
seq
monocyte chemotactic
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PCT/JP1992/000550
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English (en)
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Junichi Yamagishi
Noriyuki Matsuo
Toshikazu Fukui
Masaaki Yamada
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Dainippon Pharmaceutical Co., Ltd.
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Publication of WO1992019737A1 publication Critical patent/WO1992019737A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon

Definitions

  • the present invention relates to a process for producing a monocyte chemotactic factor polypeptide which is expected as a medicine for treating bacterial infectious diseases, improving immune functions, treating malignant tumor or the like.
  • a monocyte chemotactic factor shows an activity to attract and activate the monocyte/macrophage which plays an important role in response to inflammation and immune reaction in defence mechanism of living bodies and is highly expected of clinical applications as a medicine for treating bacterial infectious diseases, improving immune functions, treating malignant tumors or the like.
  • a monocyte chemotactic factor was prepared from the culture supernatant of phytohe aglutinin (PHA)- stimulated human peripheral blood leukocytes or the like [Yoshimura, T. et al, J. Immunol., 142, 1956-1962, 1989; Robinson, E.A. et al, Proc. Natl. Acad. Sci. USA, 86, 1850-1854, 1989; Matsushima, K. et al, J. Exp. Med., 169, 1485-1490, 1989; Decock, B. et al , Biochem. Biophys. Res.
  • PHA phytohe aglutinin
  • the present inventors have found that the monocyte chemotactic factor can be massively prepared in an insoluble form within the transformed cells by using an improved expression plasmid for highly efficient production and a selected host strain of E. coli.
  • the process of the present invention consists in using (1) a selected host strain of E. coli and (2) an expression plasmid to produce a monocyte chemotactic factor highly efficiently (3) in a high yield (4) as an unfused polypeptide (5) in an insoluble form within the cells, and it enables to provide massively said polypeptide at low costs as an economical and efficient mass production.
  • An object of the present invention is to provide a process for the production of a monocyte chemotactic factor polypeptide having monocyte chemotactic activity consisting of an amino acid sequence represented by SEQ ID N0:1 or an amino acid sequence which has truncated 1-6 N- terminal amino acids by applying recombinant DNA technology using as a host a selected E. coli or a mutant strain thereof which is transformed with an expression plasmid for highly efficient production.
  • Another object of the present invention is to provide a selected E coli or a mutant strain thereof which is transformed with an expression plasmid for highly efficient production.
  • the present invention relates to an efficient process for producin a polypeptide having monocyte chemotactic activity and its transformant which comprises using an expression plasmid having a translation termination codon and a terminator sequence downstream of the structural gene and a translation initiation codon, a ribosome binding site sequence (hereinafter referred to as "SD sequence") and a promotor sequence derived from E. coli connected upstream of the structural gene; and using as a host E. coli, an E. coli LE392 strain, an E. coli BL21(DE3) strain, an E. coli AB1899 strain, an E. coli B/r-WP2-hcr " strain or an E.
  • SD sequence ribosome binding site sequence
  • An expression plasmid for producing the monocyte chemotactic factor can be constructed in accordance with fundamental theory on recombinant DNA technology and expression (for example, Maniatis, T. et al , Molecular Cloning, a laboratory manual; Publn. Cold spring Harbor Laboratory, 1982). Thus, it can be constructed by connecting a SD sequence, a translation initiation codon and DNA encoding the monocyte chemotactic factor with a translation termination codon downstream of an appropriate promotor sequence (for example, trp, Tac, PL, etc.), connecting a terminator sequence downstream thereof, optionally connecting an appropriate repressor gene derived from E. coli, and inserting it into appropriate vector (for example, pBR322, etc.) replicable in a host strain.
  • an appropriate promotor sequence for example, trp, Tac, PL, etc.
  • DNA encoding the monocyte chemotactic factor is known and can be prepared according to the method of Furutani, Y. et al, Biochem. Biophys. Res. Co un., 15_9, 249-255, 1989 or by chemical synthesis. Further, DNA encoding the monocyte chemotactic factor which has truncated 1-6 amino acids at the N-terminus can be constructed by deleting the corresponding DNA in according with the method for side-specific mutagenesis [for example, Kunkel. T.A. et al, Methods in Enzymol., 154, 367-382, 1987].
  • the base sequence represented by SEQ ID NO: and a base sequence inclusive of said sequence are usable, and the former sequence is preferred.
  • sequence represented by SEQ ID N0:3 is preferred as the sequence from the SD sequence to the translation initiation codon (including the translation initiation codon).
  • a tryptophan promotor operator derived from E. coli is preferred as the promotor sequence.
  • a tryptophan promotor operator repressor gene (hereinafter referred to as "trpR gene") is preferred derived from E. coli such as a base sequence represented by SEQ ID NO:4. Accordingly, as for an expression plasmid, there are preferable those having the translation termination codon and the above mentioned preferable terminator sequence downstream of DNA encoding the monocyte chemotactic factor, and the above mentioned preferable sequence from SD sequence to the translation initiation codon and the preferable promotor sequence connected upstream thereof.
  • an expression plasmid having the translation termination codon and the terminator sequence represented by SEQ ID NO:2 connected downstream of DNA encoding the monocyte chemotactic factor, and the sequence represented by SEQ ID N0:3 (sequence from the SD sequence to the translation initiation codon) and the tryptophan promotor operator derived from E. coli upstream thereof.
  • the constructed expression plasmid is introduced into a selected strain of E. coli according to the calcium chloride method [Cohen, S.N. et al. Proc. Natl . Acad. Sci. USA, 69, 2110-2114, 1972], and the transformed E. coli is cultured in a conventional manner to produce and accumulate the monocyte chemotactic factor in the cultivated cells.
  • the selected strain of E. coli usable herein illustratively includes 6 strains of E. coli as shown below and mutant strains thereof.
  • E. coli LE392 (ATCC 33572)
  • E. coli LE392 strain the E. coli LE392 strain, the E. coli B/r-WP2-hcr ⁇ strain and the E. coli AB1899 strain are preferred, and the E. coli LE392 strain is most preferred.
  • the cultivated cells are disrupted, and the pellet fraction recovered from the cell-lysate is treated with a mixture of low concentrated surfactant and a protein-denaturing agent, followed by treating with deoxyribonuclease. Thereafter, a pellet fraction containing the monocyte chemotactic factor was collected. The pellet fraction is solubilized with high concentrated protein-denaturing agent and subjected to dialysis or the like for removing the protein- denaturing agent to recover the monocyte chemotactic factor as a soluble polypeptide.
  • the transformed E. coli is cultivated, and the cultivated cells containing the monocyte chemotactic factor are disrupted.
  • the pellet fraction is recovered from the cell-lysate. This pellet fraction is washed with a mixture of low concentrated surfactant and a protein-denaturing agent (for example, 0.75 M urea containing 1% Triton X-100) and digested with deoxyribonuclease, whereby a pellet fraction containing the monocyte chemotactic factor is recovered.
  • the pellet fraction is treated with high concentrated protein-denaturing agent (for example, 6 M guanidine hydrochloride) to solubilize the monocyte chemotactic factor.
  • the monocyte chemotactic factor can be recovered in the form of a soluble polypeptide by removing the protein-denaturing agent, by dialysis or lowering the concentration of the protein-denaturing agent by dilution.
  • a highly purified preparation can be obtained by subjecting to chromatographies using heparin column and ion exchange column and gel filtration. Further, other methods such as ultrafiltration, electrophoresis, affinity chromatography using specific antibody and high-performance liquid chromatography using reverse-phase column can be effective for purification.
  • the fundamental technique for purification can be performed according to the method reported by Furuta et al. [Furuta, R. et al . , J. Biochem., 106, 436-441, 1989].
  • the monocyte chemotactic factor polypeptide is accumulated in a high content in the cells and recovered in the form of an insoluble polypeptide.
  • productivity of the monocyte chemotactic factor polypeptide consisting of 76 amino acids represented by SEQ N0:1 [hereinafter referred to as "MCF(76)"] in using the E. coli LE392 strain as a host cell was about 3 mg per 100 ml of culture broth.
  • the productivity of MCF(76) with tr ' ansformants of the E. coli P2392 strain and the E. coli BL21(DE3) strain was about 2-3 mg/100 ml (culture broth), that with transformants of the E.
  • E. coli AB1899 strain and the E. coli B/r-WP2-hcr ⁇ strain was about 1 to 2 mg/100 ml (culture broth), and that with the transformant of the E. coli C600 Ir A strain was about 0.5 to 1 mg/100 ml (culture broth).
  • the monocyte chemotactic factor accumulated in the cultivated cells was detected by SDS-polyacrylamide gel electrophoresis and the protein staining method with Coo assie Brilliant Blue-R-250 (hereinafter referred to as "CBB").
  • CBB Coo assie Brilliant Blue-R-250
  • This assay is hereinafter referred to as “electrophoresis-CBB staining method”.
  • the accumulated amount was determined dens metrically based on the protein band stained with CBB.
  • ATCC number described together with names of E. coli strains is Code No. of American Type Culture Collection (12301 Parklawn Drive, Rockville, MD 20852, U.S.A.) and ME number or JE number are Code No. of National Institute of Genetics, Genetic Stocks Research Center (Tanida 1111, Mishima-shi, Shizuoka Prefecture, 411 Japan).
  • mutant strains of E. coli are antibiotic resistant strains such as a streptomycin resistant mutant strain (hereinafter referred to as "Str r ”) and a rifamycin resistant mutant strain (hereinafter referred to as "Rif"), a mutant strain conferring requirement for thymine (hereinafter referred to as "Thy”), a mutant strain conferring mutation of recA gene [Gudas, L.J. et al . , Proc. Natl. Acad. Sci. USA, 74, 5280-5284, 1977] (hereinafter referred to as
  • mutant strains of E. coli usable in the process of the present invention include those conferring mutation to chromosome genes of these parent strains according to the method using N-methyl-N'-nitro-N-nitrosoguanidine [Miller, J.H., Experiments in Molecular Genetics, 125-129, Cold Spring Harbor Laboratory (Publisher), 1972].
  • mutant strains usable in the process of the present invention are obtained as follows: at first mutant strains are obtained by cultivating a parent strain in the presence of N-methyl-N 1 - nitro-N-nitrosoguanidine.
  • mutant strains of the E. coli LE392 strain include the E. coli P2392 strain, a lisogenic strain for P2 phage of the E. coli LE392 strain.
  • mutant strains conferring new properties to those mutant strains include in mutant strains of E.
  • the monocyte chemotactic factor can be detected by SDS-polyacrylamide gel electrophoresis and the protein staining method with CBB in the cultivated cells of the strain transformed with the expression plasmid.
  • mutant strains can be prepared in a conventional manner of these technical field [Miller, J.H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (Publisher), 1972].
  • the most preferable mutant strain used in the present invention is E. coli LE392 (Rif, RecA " ), namely, a mutant strain of the E. coli LE392 strain conferring rifamycin-resistant and recA gene mutation.
  • An E. coli strain usable in the process of the present invention is incubated at 37 °C overnight on LB medium and centrifuged. The collected cells are suspended in saline, and the suspension are spread over the surface of LB agar plate containing rifa ycin and incubated at 37°C overnight. The colonies formed on LB agar plate are suspended in saline, streaked on LB agar plate containing rifamycin and incubated at 37°C overnight, whereby an E. coli (Rif) strain is isolated as a single clone. Further an E. coli (Str r ) strain is also isolated by using LB agar plate containing streptomycin in place of LB agar plate containing rifamycin.
  • the culture of the E. coli (Rif) strain is inoculated at 5% concentration to M9 medium containing thymine, trimethoprim and L- methionine and cultivated at 37°C overnight under shaking.
  • This culture is inoculated at 5% concentration to a fresh medium having the same composition and incubated at 37°C overnight under shaking.
  • This culture is spread over LB agar plate containing thymine and incubated at 37°C overnight.
  • the colonies formed are collected, and their requirement for thymine are assayed.
  • the colony showing requirement for thymine is suspended in saline, streaked on LB agar plate containing thymine and cultivated at 37°C overnight, whereby an E.
  • coli (Rif, Thy " ) strain is isolated as a single clone. Further, an E. coli (Str r , Thy " ) strain is isolated as a single clone by using the E. coli (Str r ) strain in place of the E. coli (Rif) strain.
  • E. coli KL-16-19 strain is incubated in LB medium at 37°C overnight. This culture is inoculated at 10% concentration in LB medium and incubated at 37t..
  • the E. coli (Rif, Thy " ) strain is incubated in LB medium supplemented with thymine.
  • the culture of the E. coli KL-16-19 strain is mixed with the culture of the E. coli (Rif, Thy " ) strain and incubated at 37 ⁇ to cross a male strain, the E. coli Kl-16-19 strain with a female strain, the E. coli (Rif, Thy " ) strain. Then, the recombinant strain of which the requirement for thymine of the E.
  • an E. coli (Rif, Thy " ) strain reverted to the non- requirement for thymine is obtained by collecting the colony grown on M9 agar plate not containing thymine, and the obtained colony is streaked on LB agar plate and incubated at 37 ⁇ overnight to isolate a single clone.
  • an E. coli (Rif, RecA " ) strain is isolated by assaying sensitivity to U.V. (ultraviolet rays) of this isolated clone and selecting the clone conferring high sensitivity to U.V. because recA mutant gene derived from the E. coli KL-16-19 strain is inserted into the chromosome.
  • an E. coli (Str r , RecA") strain is also isolated by treating the E. coli (Str r , Thy " ) strain in place of the E. coli (Rif, Thy " ) strain according to the above method.
  • the productivity of the monocyte chemotactic factor polypeptide MCF(76) and a polypeptide which truncated N-terminal 1 amino acid, 3 amino acids or 6 amino acids, respectively, from MCF(76) using a mutant strain, for example, the E. coli (Rif, RecA " ) strain was equal or more than that in the case using the E. coli LE392 strain.
  • the monocyte chemotactic factor namely MCF(76)
  • a polypeptide which truncated N-terminal 1 amino acid, 3 amino acids or 6 amino acids, respectively, from MCF(76) can be efficiently produced.
  • ATP adenosine triphosphate dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate dTTP deoxythymidine triphosphate
  • Example 1 Construction (1) of an expression plasmid for producing the monocyte chemotactic factor
  • An expression plasmid pHM483 for producing the monocyte chemotactic factor MCF(76) consisting of 76 amino acids represented by SEQ ID N0:1 was constructed by the following method.
  • An expression plasmid pHMC076 (cf. Referential Example 1 shown below) was digested with restriction enzymes Spel and Sail to isolate the DNA fragment of about 0.6 kilo base pair (kbp) having the base sequence represented by SEQ ID NO:5. This DNA fragment is referred to as "MCF(SS)" fragment.
  • Recombinant phage DNA was prepared by inserting this MCF(SS) fragment between the cleavage sites of restriction enzymes Sail and Xbal in the multi-cloning sites of M13mpl9 phage vector (Takara Shuzo Co.).
  • One base (G) was inserted at the site between base No. 278 and 279 (or base No.
  • this phage was transfected to E. coli CJ236, which was incubated on 2xTY medium [1.6% tryptone, 1% yeast extract, 0.5% sodium chloride] containing uridine (lz-g/ml) and chloramphenical (20/.g/ml) at 37°C for 5 hours, and single-stranded phage DNA into which uracil was introduced was isolated from the supernatant.
  • Phosphorylated mutagenic primer in which phosphate group was added to its 5'-terminus with T4 polynucleotide kinase was allowed to hybridize with the single-stranded phage DNA containing uracil prepared above in annealing buffer [20 M Tris-HCl buffer (pH 7.4) containing 2 mM magnesium chloride and 50 mM sodium chloride] at 70 °C for 10 minutes and the mixture was gradually chilled up to 30°C at a rate of 1°C /minute to hybridize the primer to phage DNA.
  • phage DNA was allowed to react with T4 DNA polymerase in synthesis buffer [10 mM Tris- HCl buffer (pH 7.4) containing each 0.4 mM of four deoxyribonucleotide triphosphates (dGTP, dCTP, dATP and dTTP), 0.75 mM ATP, 3.75 mM magnesium chloride and 1.5 mM dithiothreitol] under reaction conditions on ice for 5 minutes, at 25 °C for 5 minutes and at 37°C for 90 minutes to synthesize complementary DNA to the template phage DNA, and the termini of the complementary DNA were ligated by T4 DNA ligase, and the reaction was stopped by freezing at -20°C in order to prepare Double- stranded closed-circular DNA.
  • synthesis buffer 10 mM Tris- HCl buffer (pH 7.4) containing each 0.4 mM of four deoxyribonucleotide triphosphates (dGTP, dCTP, dATP
  • the obtained DNA was transfected to E ⁇ coli JM105 and incubated to isolate a double-stranded replicative form of DNA in which the mutation was introduced (hereinafter referred to as "mutated double-stranded DNA”).
  • the mutated double stranded DNA was digested with restriction enzymes Dral and SaJI to isolate DNA fragment having the base sequence represented by SEQ ID N0:7. This DNA fragment is referred to as "MCF(DS)".
  • the recognition sequence (GGATCC) at base No. 248-253 of SEQ ID NO:7 with restriction enzyme Ba HI was constructed by inserting one base (G) due to the method above.
  • the expression plasmid pHMC076 was digested with restriction enzyme Dral and Sail to cut into two fragments, whereby there was isolated a larger DNA fragment (hereinafter referred to as "HMC076 vector fragment") including a tryptophan promotor sequence derived from E. coli (hereinafter referred to as "trp promotor”), an a picill in-resistant gene, and a replication origin but not including the structural gene of the monocyte chemotactic factor MCF(76).
  • the HMC076 vector fragment was ligated with T4 DNA ligase to MCF (DS) fragment to construct an expression plasmid pHMC076-B.
  • the expression plasmid pHMC076-B was digested with restriction enzymes Xhol and BamHI to remove 5 bases (TCGAG, fragment corresponding to base No. 244-248 of the base sequence represented by SEQ ID N0:7) between the cleavage sites of Xhol and BamHI, and a double-stranded DNA prepared by annealing two chemically synthesized DNAs represented by SEQ ID N0:8 and 9 was integrated in this site to construct an expression vector pHM483.
  • the sequence of this DNA includes the base sequence represented by SEQ ID N0:2 and was set so as to function as a terminator, namely signal for terminating transcription.
  • the base sequence was determined according to the dideoxy method using 7-DEAZA sequencing kit (Takara Shuzo Co. ).
  • Expression plasmids for producing the monocyte chemotactic factor which truncated N-terminal 1-6 amino acids from MCF(76) were constructed as follows. Polypeptides which truncated N-terminal 1 amino acid, 3 amino acids and 6 amino acids from MCF(76) are referred to as "MCF(75), MCF(73) and MCF(70)", "respectively. Expression plasmids for producing these 3 polypeptides which truncated N-terminal amino acids are referred to as "pHM484, pHM485 and pHM486", respectively in that order.
  • An expression plasmid pH484 for producing the monocyte chemotactic factor MCF(75) was constructed by the following method.
  • the expression plasmid pHM483 obtained by the method obtained in Example 1 was digested with restriction enzymes S_pel and SaJI to isolate DNA fragment containing the region encoding MCF(76). This DNA fragment was inserted between the cleavage sites of restriction enzyme Sail and Xbal in the multi-cloning sites of M13mpl9 phage vector to construct recombinant phage DNA(MCF76).
  • the codon (CAG) corresponding to the N-terminal amino acid (Gin) of MCF(76) was deleted according to the site-specific mutagenic method described in Example 1.
  • As a mutagenic primer was used chemically synthesized DNA having the base sequence represented by SEQ ID N0:11.
  • the resultant mutated double-stranded DNA was digested with restriction enzymes Dral and Xhol to isolate the mutated DNA fragment which truncated the codon corresponding to the N-terminal amino acid (Gin) of MCF(76).
  • the expression plasmid pHM483 constructed by the method as described in Example 1 was digested with restriction enzymes Dral and Xhol to cut into two fragments to isolate a larger DNA fragment (hereinafter referred to as "HM483 vector fragment") including the trp promotor, the terminator sequence, the ampicill in-resistant gene, and the replication origin but not including the structural gene of the monocyte chemotactic factor MCF(76).
  • This HM483 vector fragment was ligated to said mutated DNA fragment with T4 DNA ligase to construct the expression plasmid pHM484.
  • the base sequence of the expression plasmid pHM484 corresponded to the sequence which truncated the codon corresponding to the N-terminal amino acid (Gin) of MCF(76) from the base sequence of the expression plasmid pHM483.
  • the base sequence was determined by the dideoxy method using 7-DEAZA sequence kit.
  • Expression plasmids pHM485 and pHM486 for producing the polypeptide namely MCF(73) and MCF(70) which truncated N-terminal 3 amino acids and 6 amino acids from MCF(76) were constructed with recombinant phage DNA(MCF76) and the respectively corresponding mutagenic primers according to the method described in above section (1).
  • mutagenic primers were used the respective chemically synthesized DNA corresponding to the base sequence encoding the peptide to be truncated.
  • An expression plasmid pHM485 was constructed as follows: the mutated double-stranded DNA which truncated the base sequence (CAGCCAGAT) encoding the N-terminal 3 amino acids (GlnProAsp) of MCF(76) was obtained by using recombinant phage DNA(MCF76) and a mutagenic primer represented by SEQ ID NO:12 according to the site-specific mutagenic method. Then, the mutated DNA fragment obtained by digesting the mutated double-stranded DNA with restriction enzyme Dral and Xhol was ligated to said HM483 vector fragment.
  • An expression plasmid pHM486 was constructed as follows: the mutated double-stranded DNA which truncated the base sequence (CAGCCAGATGCAATCAAT) encoding N-terminal 6 amino acids (GlnProAspAlalleAsn) of MCF(76) was obtained by using recombinant phage DNA(MCF76) and a mutagenic primer represented by SEQ ID NO:13 according to the site-specific mutangenesis. Then, the mutated DNA fragment obtained by digesting the mutated double-stranded DNA with restriction enzymes Dral and Xhol was ligated to HM483 vector fragment.
  • An expression plasmid pHM583 for producing the monocyte chemotactic factor MCF(76) in which trpR gene had been inserted was constructed by the following method.
  • Chromosomal DNA was extracted from E. coli HB101 according to the following method as modified from Cosloy et al., Mol. Gen. Genet., 124, 1-10, 1973.
  • the cultivated cells were suspended in 0.1 M sodium chloride solution containing 0.1 M disodiu ethylenediamine- tetraacetate solution, mixed with 50 mM Tris-HCl buffer (pH 8.0) containing pronase K (50 -g/ml) and 0.5% SDS and heated at 42 °C for 1 hour to dissolve the cells and digest the bacterial protein.
  • the resulting chromosomal DNA was digested with restriction enzyme NlalV to isolate about lkbp fragment according to the low-melting- temperature agarose .gel electrophoresis method. This DNA fragment was inserted between the cleavage site of restriction enzyme Smal in the multi-cloning site of M13mpl8 phage vector to prepare recombinant phage DNA library.
  • DNA fragment including trpR gene represented by ?EQ ID NO:15 was cloned with a probe having the base sequence (corresponding to the sequence from base No. 363 to 376 of SEQ ID N0:15) represented by SEQ ID N0:14 from this library according to the following method as modified from the plaque-hybridization method [Sambrook, J. et al., Molecular Cloning, a Laboratory Manual, Vol. 1, 2.108, Cold Spring Harbor Laboratory Press (Publisher), 1989].
  • said recombinant phage DNA library was at first infected to E. coli JM105 to form plaques. Then, about 500 single plaques were individually infected to E. coli JM105, and the strains infected were incubated to recover the resulting phage.
  • prepared phages were dot-blotted to nitrocellulose filters, which were immersed in 1.5 M sodium chloride solution containing 0.1N sodium hydroxide, allowed to stand still at room temperature for 5 minutes and neutralized by soaking in 0.5 M Tris-HCl buffer (pH 7.0) containing 3 M sodium chloride at room temperature for 10 minutes. This filter was baked at 80 * 0 for 2 hours to fix phage DNA thereon.
  • the filter on which said recombinant phage DNA was fixed was allowed to incubated at about 40 ⁇ for 48 hours in 5 fold-concentrated SSC (a solution of 0.15 M sodium chloride containing 0.015 M sodium citrate is referred to as "SSC") containing 32 P-labeled probe (about 1,000,000 cpm/ml), denatured salmon sperm DNA (100/- g/ml), 0.1% SDS, 1 mM disodium ethylenediamine tetraacetate, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and 0.02% ficoll.
  • SSC 5 fold-concentrated SSC
  • the filtrate was heated at 65O for 1 hour in 5-fold- concentrated SSC containing 0.1% SDS and washed 3 times with 2 fold- concentrated SSC.
  • the filter was dried and subjected to autoradiography to detect the phage which was bound to 32 P-labeled probe.
  • a recombinant phage corresponding to this phage DNA is referred to as "candidate phage”.
  • the candidate phage was obtained at a frequency of about one per 100 phages from the recombinant phage DNA library.
  • the cleavage recognition sequence (GAATTC) of restriction enzyme EcoRI was inserted between base No. 116 and base No. 117 of SEQ ID NO:15
  • the cleavage recognition sequence (GGATCC) of restriction enzyme BamHI was inserted between base No. 956 and base No. 957 of SEQ ID N0:15 according to the site-specific mutagenesis method as described in Example 1.
  • the resultant mutated double-chain DNA was digested with restriction enzymes EcoRI and BamHI to isolate trpR gene fragment (SEQ ID N0:4) having the sequence of the cohesive end of EcoRI at the 5'-end of the sequence of base No. 117 to 956 of SEQ ID NO:15 and having the sequence of the cohesive end of BamHI at the 3'-end.
  • This trpR gene fragment is referred to as "TRP(EB) fragment".
  • the expression plasmid pHM483 constructed by the method as described in Example 1 was digested with restriction enzymes EcoRI and BamHI to cut into two fragments, whereby a larger DNA fragment including the trp promotor, the terminator sequence, the a picillin resistant gene, the structural gene of the monocyte chemotactic factor, and the replication origin was isolated.
  • This DNA fragment is referred to as HM483(EB) vector fragment.
  • Said TRP(Ei-) fragment was ligated this vector fragment with T4 DNA ligase to construct the expression plasmid pHM583.
  • the base sequence of the expression plasmid pHM583 was corresponded to the sequence which deleted 6 bases (AATTCG, the fragment corresponding to base No.
  • Expression plasmids for producing the monocyte chemotactic factors MCF(75), MCF(73) and MCF(70) into which trpR gene was inserted were constructed by the following method. These 3 expression plasmids are referred to as "pHM584, pHM585 and pHM586", respectively in that order.
  • These three vector fragments were ligated TRP(EB) fragment as prepared by the method of Example 3 with T4 DNA ligase to construct expression plasmids pHM584, pHM585 and pHM586, respectively.
  • the base sequence of these expression plasmids were determined by the dideoxy method using 7-DEAZA Sequence Kit.
  • the monocyte chemotactic factor MCF(76) was prepared by introducing the expression plasmid PHM483 into the E. coli LE392 strain according to the following method.
  • the E. coli LE392 strain was inoculated to LB medium [1% tryptone, 0.5% yeast extract, 1% sodium chloride (pH 7.2)] and incubated at 30 °C until the OD 600 nm reached about 0.5. This culture was allowed to stand still for 30 minutes on ice and centrifuged to obtain the cells. The cells were suspended in 50 mM calcium chloride solution, allowed to stand still on ice for 60 minutes and centrifuged to obtain the cells. The cells were suspended in 50 mM calcium chloride solution containing 20% glycerin.
  • E. coli LE392/ .-iM483 The resultant transformed E. coli is referred to as "E. coli LE392/ .-iM483".
  • E. coli LE 392/pHM483 was incubated at 37°C overnight on LB medium, and the culture was inoculated at 1% concentration to a productive medium [composition: 1.5% disodium phosphate 12 hydrate, 0.3% onopotassium phosphate, 0.1% ammonium chloride, 2// g/ml vitamin Bi , 0.5% Casamino acid, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 1% tryptone, 0.5% yeast extract, 1% sodium chloride, and 0.4% glycerin], added with 3-indoleacrylic acid to a final concentration of 20 - g/ml, and incubated at 37°C for about 24 hours.
  • a productive medium Composition: 1.5% disodium phosphate 12 hydrate, 0.3% onopotassium phosphate, 0.1% ammonium chloride, 2// g/ml vitamin Bi , 0.5% Casamino acid, 2 mM magnesium sulfate, 0.1 m
  • the cells collected by centrifugation were suspended in 20 mM Tris-HCl buffer (pH 7.5) and then disrupted by sonification to obtain a cell-lysate.
  • the content of MCF(76) in this cell-lysate was assayed by the electrophoresis-CBB staining method.
  • MCF(76) a large amount of MCF(76) was detected in the cell- lysate, and most of MCF(76) was recovered as an insoluble polypeptide in the pellet fraction after centrifugation of the cell-lysate.
  • the content of MCF(76) in the total insoluble protein of the culture of E. coli LE392/pHM483 was not less than about 20%, and the productivity of MCF(76) was equivalent to about 3 mg per 100 ml culture.
  • a methionine residue due to the translation initiation codon and the methionine residue was found to have hardly been removed.
  • the monocyte chemotactic factor MCF(76) was prepared by incubating each transformant, made by introduction of the expression plasmid pHM483 constructed by the method of Example 1 according to the method of Example 5, using the E. coli P2392 strain, the E. coli BL21(DE3) strain, the E. coli AB1899 strain, the E. coli B/r-WP2-hcr " strain and the E. col C600 hflA strain as a host cell.
  • a large amount of MCF(76) was observed by the electrophoresis-CBB staining method in the cell-lysate of the transformants of the E. coli P2392 strain, the E. coli BL21(DE3) strain, the E.
  • E. coli AB1899 strain the E. coli B/r-WP2-hcr " strain and the E. coli C600 hflA strain. Its content was equivalent to about 2-3 mg/100 ml (culture broth) in the transformants of the E. coli P2392 strain and the E. coli BL21(DE3) strain, about 1-2 mg/100 ml (culture broth) in the transformants of the E. coli AB1899 strain and the E. coli B/r-WP2-hcr ⁇ strain, and about 0.5-1 mg/100 ml (culture broth) in the E. coli C600 hflA strain.
  • Example 7 Production (1) of the monocyte chemotactic factor in the E. coli LE392 (Rif, RecA " ) strain as a host cell
  • the monocyte chemotactic factor MCF(76) was prepared in the direct expression system, using the E. coli LE392 (Rif, RecA " ) strain as a host cell made by the method of Referential Example 2 described below.
  • the expression plasmid pHM483 constructed by the method of Example 1 was introduced into the E. col i LE392 (Rif, RecA " ) strain according to the method of Example 5, and the resultant transformed E. coli was incubated. It was confirmed that MCF(76) could be equally produced in the direct expression system, using the E. coli LE392 (Rif, RecA " ) strain as a host cell, compared with the case using E.
  • Example 8 Production (2) of the monocyte chemotactic factor in the E. coli LE392 (Rif, RecA " ) strain as a host cell
  • the monocyte chemotactic factors MCF(75), MCF(73) and MCF(70), namely N-terminal truncated polypeptide of MCF(76) were prepared in the direct expression system, using the E. coli LE392 (Rif, RecA " ) strain made by the method of Referential Example 2 described below as a host cell.
  • expression plasmids pHM484, pHM485 or pHM486 for producing each truncated polypeptide constructed by the method of Example 2 was introduced into the E. coli LE392 (Rif, RecA " ) strain accordi ' i to the method of Example 5, and the resultant transformed E. coli were incubated.
  • Example 9 Production (3) of the monocyte chemotactic factors using the E. coli LE392 (Rif, RecA " ) strain as a host cell
  • the monocyte chemotactic factor MCF(76) and N-terminal truncated polypeptides were prepared in the direct expression system using the E. coli LE392 (Rif, RecA " ) strain as a host cell prepared in Referential Example 2 as described below.
  • expression plasmids for producing the various monocyte chemotactic factors in which trpR gene inserted by the method described in Example 3 and Example 4 were introduced into the E. coli LE392 (Rif, RecA " ) strain according to the method of Example 5, and each transformed E. coli was cultivated. It was confirmed that said polypeptide was equally or more produced in the transformed E. coli LE392 (Rif, RecA " ), compared with the case using the E. coli LE392 strain, by assaying the productivity of each monocyte chemotactic factor in each cultivated cell according to the electrophoresis-CBB staining method.
  • An expression plasmid pHMC076 for producing the monocyte chemotactic factor MCF(76) was constructed by the following method.
  • DNA encoding MCF(76) was prepared by using a recombinant plasmid pHMCF7 [Furutani, Y. et al , Biochem. Biographys. Res. Co mun., 159, 249-255, 1989].
  • the base sequence of cDNA inserted in a recombinant plasmid pHMCF7 was shown by SEQ ID NO:18.
  • the recombinant plasmid pHMCF7 was digested with restriction enzyme P_stl to isolate DNA fragment including the base sequence encoding MCF(76).
  • This DNA fragment was inserted into the cleavage site of restriction enzyme PstI in the multi-cloning site of M13mpl8 phage vector (Takara Shuzo Co.).
  • the base sequence represented by SEQ ID NO:21 was inserted upstream of the codon (CAG) corresponding to the N-terminal amino acid (Gin) of MCF(76), using this recombinant phage DNA and two chemically synthesized DNAs having the base sequence represented by SEQ ID N0:19 and 20 as each mutagenic primer, according to the site-specific mutagenesis method described by Example 1, and further the base sequence of 5' -TGACTCGAG-3 1 was inserted downstream of the termination codon continuing to the C- terminal amino acid (Thr).
  • the resultant mutated double-stranded DNA is referred to as "(DraI)(XhoI) inserted phage DNA".
  • the partial base sequence is shown in SEQ ID N0:22.
  • This mutated double-stranded DNA was digested with restriction enzymes Dral and Xhol to isolate DNA fragment corresponding to the 142-384th of the base sequence represented by SEQ ID N0:22.
  • This DNA fragment is referred to as "MCF (Dral-Xhol) fragment”.
  • This MCF (Dral-Xhol) fragment included the sequence having the translation initiation codon upstream of DNA encoding MCF(76) and the translation termination codon downstream thereof.
  • an expression vector pEP205 [Furuta, R. et al., J. Biochem., 106, 436-441, 1989] was digested with restriction enzymes Dral and Xhol to isolate a large DNA fragment completely free of human IL-l ⁇ r structural gene inserted in pEP205. This DNA fragment is referred to as EP205 vector fragment.
  • the base sequence of EP205 vector fragment includes the trp promotor, the ampicillin-resistant gene and the replication origin.
  • Said MCF (Dral-Xhol) fragment was ligated to EP205 vector fragment with T4 DNA ligase to construct the expression plasmid pHMC076. The base sequence was determined by the dideoxy method using 7-DEAZA sequence kit.
  • the structure of the expression vector pEP205 was disclosed in Fig. 2 (p. 437) of Furuta, R. et al. report [J. Biochem., 106, 436-441, 1989].
  • This EP205 vector fragment corresponds to about 3.7 kbp of (d)-(a)-(b) region except for (c) region in the structure of Fig. 2.
  • the (a)-(b) region includes the trp promotor and the SD sequence and is referred to as "E. coli trp-SD sequence".
  • the base sequence is represented by SEQ ID N0:23.
  • the (d) region is due to pBRS6 [Ya ada, M. et al., J.
  • E. coli LE392 (Rif) a mutant strain having rifamycin resistance (Rif)
  • a mutant strain having rifamycin resistance (Rif)
  • a mutant strain having rifamycin resistance (Rif)
  • a mutant strain having rifamycin resistance (Rif)
  • a mutant strain having rifamycin resistance (Rif)
  • a mutant strain having rifamycin resistance (Rif)
  • E. coli LE392 (Rif, Thy " ) conferring requirement for thymine
  • a recA mutant strain hereinafter referred to as "E. coli LE392 (Rif, RecA " )”
  • U.V. high sensitivity by integrating recA mutant gene a mutant strain having rifamycin resistance and U.V. high sensitivity by integrating recA mutant gene.
  • the E. coli LE392 strain was incubated at 37°C overnight under shaking in 10 ml of LB medium, and the cells collected by centrifugation were suspended in 1 ml of saline.
  • the cell suspension (0.1 ml) was spread over LB agar plate containing rifamycin (100. g/ml) and incubated at 37°C overnight.
  • the colony formed on LB agar plate was suspended in saline, streaked on LB agar plate containing rifamycin (100 / g/ml) and incubated at 37°C overnight to isolate single colonies.
  • the requirement for methionine was determined using an index of growing on M9 agar medium [composition: 1.5% disodium phosphate 12 hydrate, 0.3% monopotassium phosphate, 0.1% ammonium chloride, 2 ⁇ g/ml vitamin Bi , 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.05% sodium chloride, 0.2% glucose and 1.5% agar] containing L-methionine (40/- g/ml) but not growing on M9 agar medium free of L- methionine.
  • the resultant E. coli LE392 (Rif) strain was incubated on LB medium containing rifamycin (100// g/ml), and this culture was mixed with dimethyl sulfoxide (DMSO) at 7% concentration and stored by freezing.
  • DMSO dimethyl sulfoxide
  • the culture of the E. coli LE392 (Rif) strain obtained by incubating overnight under shaking on LB medium containing rifamycin (100/ g/ml) was inoculated at 5% concentration to M9 medium [composition: 1.5% disodium phosphate 12 hydrate, 0.3% monopotassium phosphate, 0.1% ammonium chloride, 2 g/ml vitamin Bi, 2 mM magnesium sulfate, 0.1 M calcium chloride, 0.05% sodium chloride and 0.2% glucose] containing thymine (200// g/ml), trimethoprim (200// g/ml) and L- methionine (40 / g/ml) and incubated at 37O overnight under shaking.
  • M9 medium Composition: 1.5% disodium phosphate 12 hydrate, 0.3% monopotassium phosphate, 0.1% ammonium chloride, 2 g/ml vitamin Bi, 2 mM magnesium sulfate, 0.1 M calcium chloride,
  • the culture was inoculated at 5% concentration to fresh M9 medium having the same composition and incubated at 37 ⁇ overnight under shaking.
  • the resultant culture was spread over LB agar plate containing thymine (200 / g/ml) and incubated at 370. Colonies formed were assayed on requirement for thymine.
  • the requirement for thymine was determined with an index that allowed to grow on the M9 agar plate containing thymine (200// g/ml) and L-methionine (40 g/ml) but didn ' t allow to grow on the M9 agar plate containing L-methionine (40/_ g/ml).
  • coli LE392 (Rif, Thy " ) strain prepared was incubated on LB medium containing rifamycin (100// g/ml) and thymine (200 / g/ml ), and the resultant culture was mixed with dimethyl sulfoxide at 7% concentration and stored by freezing.
  • the E. coli LE392 (Rif, Thy " ) strain was incubated on LB medium containing thymine (200 / g/ml) under the same conditions as described above to give a culture having the same level of cell number.
  • the resultant culture of the E. coli KL-16-19 strain was mixed with the culture of the E. coli LE392 (Rif, Thy " ) strain at a ratio of 1:10 and incubated at 37°C under shaking very slowly to cross a male strain, the E. coli KL-16-19 strain with a female strain, the E. coli LE392 (Rif, Thy " ) strain.
  • this mixed culture was chilled on ice, spread over M9 agar plate containing rifamycin (100// g/ml) and L-methionine (40// g/ml) and incubated at 37°C for 2 days.
  • the colony which grew over M9 agar plate free of thymine was selected to obtain a recombinant strain of the E. coli LE392 (Rif, Thy " ) strain of which requirement for thymine was reverted to non-requirement.
  • This strain was suspended in saline, streaked over LB agar plate and incubated at 37°C overnight to isolate single clones. It was again confirmed that one of the single clones isolated showed requirement for methionine and rifamycin resistance but free of requirement for thymine. Assays for these properties were as shown above.
  • sensitivity to U.V. of the clone isolated was assayed, whereby the clone showing high sensitivity to'u.V. by integrating recA mutant gene derived from the E. coli KL-16-19 strain was selected.
  • Assay for sensitivity to U.V. was performed by inoculating a strain on LB agar plate, irradiating U.V. (0.5 rnW/crn 2 ) for 5 seconds, incubating at 37 °C overnight and deciding whether it grew or not. Any strain showing high sensitivity to U.V. was selected, using criteria that it didn't grow under irradiation of U.V. as described above under which conditions the E. coli LE392 strain, the E.
  • E. coli LE392 (Rif) strain or the E. coli LE392 (Rif, Thy " ) strain were able to grow.
  • the resultant strains showing high U.V. sensitivity are referred to as "E. coli LE392 (Rif, RecA " ) strain.
  • E. coli LE392 (Rif, RecA " ) strain prepared was incubated on LB medium containing rifamycin (100// g/ml), mixed with dimethyl sulfoxide at 7% concentration and stored by freezing.
  • E. coli B/r-WP2-hcr ⁇ As a parent strain, an E. coli B/r- WP2-hcr ⁇ (Rif) strain showing rifamycin resistance and an E. coli B/r- WP2-hcr ⁇ (Rif, Thy " ) strain showing rifamycin resistance and requirement for thymine were prepared according to the method described in Referential Example 2.
  • E. coli B/r-WP2-hcr ⁇ strain As a parent strain, an E_ coli B/r-WP2-hcr " (Str r ) strain showing streptomycin resistance and an E. coli B/r-WP2-hcr " (Str r , Thy) strain showing streptomycin resistance and requirement for thymine were prepared according to the method described in Referential Example 2. In this case, streptomycin (100 / g/ml) was used in place of rifamycin in each step for preparing mutant strains.
  • MOLECULE TYPE other nucleic acid sequence from SD sequence to translation initiation codon FEATURE
  • GGC GAA ATG AGC CAG CGT GAG TTA AAA AAT GAA CTC GGC GCA GGC ATC 482 Gly Glu Met Ser Gin Arg Glu Leu Lys Asn Glu Leu Gly Ala Gly He
  • SEQ ID NO:14 SEQUENCE LENGTH:14 SEQUENCE TYPE:nucleic acid STRANDEDNESS:single TOPOLOGY:linear
  • TISSUE TYPE human promyelocytic leukaemia cells
  • GCCTCCAGC ATG AAA GTC TCT GCC GCC CTT CTG TGC CTG CTG CTC ATA GCA 111 Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu He Ala -20 -15 -10
  • GCCTCCAGC ATG AAA GTC TCT GCC GCC CTT CTG TGC CTG CTG CTC ATA GCA 111 Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu He Ala -20 --15 -10
  • AAG CAA ACC CAA ACT CCG AAG ACT TGATGACTCG AGACACTCAC TCCACAACCC 407

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Abstract

Procédé de préparation d'un facteur chimiotactique de monocyte, qui consiste à produire par recombinaison un plasmide d'expression dans lequel a été inséré un ADN codant un facteur chimiotactique de monocyte composé de 76 aminoacides ou un polypeptide de ce facteur, qui présente des aminoacides tronqués de terminaison N, une séquence terminateur étant insérée dans le plasmide en aval dudit ADN, à introduire le plasmide d'expression dans un E. coli sélectionné, tel qu'une souche LE392 d'E. coli ou analogue, afin de transformer celui-ci, et à cultiver ledit E. coli transformé.
PCT/JP1992/000550 1991-05-09 1992-04-27 Procede de preparation d'un polypeptide de facteur chimiotactique de monocyte et souche utilisee pour cette preparation WO1992019737A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038559A1 (fr) * 1995-05-31 1996-12-05 Dana Farber Cancer Institute Mutations de deletion de l'extremite n de chemokine
US5750652A (en) * 1994-01-21 1998-05-12 Yale University Deltex proteins
WO1999005279A1 (fr) * 1997-07-25 1999-02-04 Zeneca Limited Analogues de mcp-1
US7396652B2 (en) 1995-02-17 2008-07-08 Incyte Corporation Chemokine PANEC-2 polypeptides and compositions and methods related thereto
US7713521B2 (en) 2005-08-12 2010-05-11 Schering Corporation MCP1 fusions
US8524217B2 (en) 2010-05-11 2013-09-03 Merck Sharp & Dohme Corp. MCP1-Ig fusion variants

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1990007863A1 (fr) * 1989-01-01 1990-07-26 The United States Of America, Represented By The Secretary, United States Department Of Commerce Polypeptide possedant une activite de facteur chimiotactique monocyte humain et adn codant ce polypeptide

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Publication number Priority date Publication date Assignee Title
WO1990007863A1 (fr) * 1989-01-01 1990-07-26 The United States Of America, Represented By The Secretary, United States Department Of Commerce Polypeptide possedant une activite de facteur chimiotactique monocyte humain et adn codant ce polypeptide

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 159, no. 1, 28 February 1989, DULUTH, MINNESOTA US pages 249 - 255; YASUJI FURUTANI ET AL: 'Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor;MCAF' cited in the application *
GHERNA R., PIENTA P. & COTE R. 'ATCC , CATALOGUE OF BACTERIA AND PHAGES .17th edition' 1989 , ATCC , ROCKVILLE, US *
JOURNAL OF BIOCHEMISTRY. vol. 106, 1989, TOKYO JP pages 436 - 441; RYUJI FURUTA ET AL: 'Production and characterization of recombinant human neutrophil chemotactic factor' cited in the application *
JOURNAL OF MOLECULAR BIOLOGY vol. 189, no. 1, 5 May 1986, LONDON ;GB. pages 113 - 130; F. W. STUDIER ET AL: 'Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes' cited in the application *
NUCLEIC ACIDS RESEARCH. vol. 9, 1981, ARLINGTON, VIRGINIA US pages 6647 - 6667; C. YANOFSKY AT AL: 'The complete nucleotide sequence of the tryptophan operon of E. coli' *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854412A (en) * 1993-11-12 1998-12-29 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5705360A (en) * 1993-11-12 1998-01-06 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5739103A (en) * 1993-11-12 1998-04-14 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5750652A (en) * 1994-01-21 1998-05-12 Yale University Deltex proteins
US7396652B2 (en) 1995-02-17 2008-07-08 Incyte Corporation Chemokine PANEC-2 polypeptides and compositions and methods related thereto
US7803373B2 (en) 1995-02-17 2010-09-28 Incyte Pharmaceuticals, Inc. Chemokine panec-1 antibodies
US8617551B2 (en) 1995-02-17 2013-12-31 Incyte Corporation Methods of diagnosing pancreatic inflammation by using chemokine PANEC-1 antibodies
US9090687B2 (en) 1995-02-17 2015-07-28 Incyte Corporation Methods of treating pancreatitis
EP1647597A1 (fr) * 1995-05-31 2006-04-19 Dana Farber Cancer Institute Mutations de deletion de l'extremite N de chemokine
WO1996038559A1 (fr) * 1995-05-31 1996-12-05 Dana Farber Cancer Institute Mutations de deletion de l'extremite n de chemokine
WO1999005279A1 (fr) * 1997-07-25 1999-02-04 Zeneca Limited Analogues de mcp-1
US6383782B1 (en) 1997-07-25 2002-05-07 Zeneca Limited MCP-1 analogs
US7713521B2 (en) 2005-08-12 2010-05-11 Schering Corporation MCP1 fusions
US7972591B2 (en) 2005-08-12 2011-07-05 Schering Corporation Methods for treating rheumatoid arthritis and multiple sclerosis using MCP1 fusions
US8282914B2 (en) 2005-08-12 2012-10-09 Merck, Sharp & Dohme Corp. Method for treating atherosclerosis by administering human MCP1 fusions
US8524217B2 (en) 2010-05-11 2013-09-03 Merck Sharp & Dohme Corp. MCP1-Ig fusion variants

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