Home LiteratureArticle Details
PMID: 3889908 Published · ppublish English Journal Article

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 82 ·No. 11 ·1985-06-00 ·Pages 3543-7

Mandecki W, Mollison KW, Bolling TJ, Powell BS, Carter GW, Fox JL

Abstract

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.

MeSH Terms
Animals Base Sequence Biological Assay Cloning, Molecular Complement C5/genetics,pharmacology Complement C5a DNA/chemical synthesis Escherichia coli/genetics Gene Expression Regulation Guinea Pigs Humans Molecular Weight Muscle Contraction/drug effects Oligonucleotides/chemical synthesis Operon
Chemicals
Complement C5 Oligonucleotides Complement C5a DNA
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Mandecki W
Mollison K W
Bolling T J
Powell B S
Carter G W
Fox J L
References (42)
42 references, click to expand
  1. The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement.
    J Exp Med. 1968 Feb 1;127(2):371-86 PMID: 4383923
  2. Characterization of translational initiation sites in E. coli.
    Nucleic Acids Res. 1982 May 11;10(9):2971-96 PMID: 7048258
  3. Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase.
    J Clin Invest. 1970 Dec;49(12):2427-36 PMID: 4098172
  4. Mechanisms of lysosomal enzyme release from human leukocytes: microtubule assembly and membrane fusion induced by a component of complement.
    Proc Natl Acad Sci U S A. 1973 Oct;70(10):2916-20 PMID: 4355374
  5. The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli.
    Proc Natl Acad Sci U S A. 1973 Dec;70(12):3585-9 PMID: 4587256
  6. Activation, activities and pharmacologically active products of complement.
    Pharmacol Rev. 1974 Jun;26(2):125-69 PMID: 4609350
  7. Human C3 and C5: subunit structure and modifications by trypsin and C42-C423.
    J Immunol. 1975 Feb;114(2 pt 2):815-22 PMID: 1112981
  8. Complement-mediated release of histamine from human leukocytes.
    J Immunol. 1975 Mar;114(3):1101-6 PMID: 46261
  9. Determinant of cistron specificity in bacterial ribosomes.
    Nature. 1975 Mar 6;254(5495):34-8 PMID: 803646
  10. Release of histamine from rat mast cells by the complement peptides C3a and C5a.
    Immunology. 1975 Jun;28(6):1067-80 PMID: 48505
  11. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    Anal Biochem. 1976 May 7;72:248-54 PMID: 942051
  12. Partial characterization of human C5a anaphylatoxin. I. Chemical description of the carbohydrate and polypeptide prtions of human C5a.
    J Immunol. 1976 Nov;117(5 Pt 1):1688-94 PMID: 1002996
  13. Complement (C5-a)-induced granulocyte aggregation in vitro. A possible mechanism of complement-mediated leukostasis and leukopenia.
    J Clin Invest. 1977 Jul;60(1):260-4 PMID: 874088
  14. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin.
    Science. 1977 Dec 9;198(4321):1056-63 PMID: 412251
  15. Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions.
    J Immunol. 1978 Jan;120(1):109-15 PMID: 342601
  16. Primary structural analysis of the polypeptide portion of human C5a anaphylatoxin. Polypeptide sequence determination and assignment of the oligosaccharide attachment site in C5a.
    J Biol Chem. 1978 Oct 10;253(19):6955-64 PMID: 690134
  17. Chemical synthesis and cloning of a gene for human beta-urogastrone.
    Nucleic Acids Res. 1982 Aug 11;10(15):4467-82 PMID: 6290982
  18. Codon usage in bacteria: correlation with gene expressivity.
    Nucleic Acids Res. 1982 Nov 25;10(22):7055-74 PMID: 6760125
  19. Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.
    Nucleic Acids Res. 1983 Mar 25;11(6):1707-23 PMID: 6300774
  20. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.
    Microbiol Rev. 1983 Mar;47(1):1-45 PMID: 6343825
  21. Chemical synthesis and biological studies on mutated gene-control regions.
    Cold Spring Harb Symp Quant Biol. 1983;47 Pt 1:411-8 PMID: 6345060
  22. Role of cell surface contact in the kinetics of superoxide production by granulocytes.
    J Clin Invest. 1983 Jul;72(1):113-21 PMID: 6308042
  23. A specific enzyme-linked immunosorbent assay (ELISA) for the determination of human C5a antigen.
    J Immunol Methods. 1983 Sep 16;62(3):305-14 PMID: 6193199
  24. Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli.
    Nucleic Acids Res. 1983 Sep 24;11(18):6419-35 PMID: 6312423
  25. Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region.
    Gene. 1984 Nov;31(1-3):263-7 PMID: 6396162
  26. Anaphylatoxins: C3a and C5a.
    Adv Immunol. 1978;26:1-53 PMID: 358800
  27. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters.
    J Mol Biol. 1978 Nov 15;125(4):467-90 PMID: 368341
  28. Expression in Escherichia coli of chemically synthesized genes for human insulin.
    Proc Natl Acad Sci U S A. 1979 Jan;76(1):106-10 PMID: 85300
  29. Antibiotic uptake by alveolar macrophages.
    J Lab Clin Med. 1980 Mar;95(3):429-39 PMID: 7354244
  30. Amino acid sequence of the anaphylatoxin from the fifth component of porcine complement.
    J Biol Chem. 1980 May 25;255(10):4710-5 PMID: 7372604
  31. Rapid DNA isolations for enzymatic and hybridization analysis.
    Methods Enzymol. 1980;65(1):404-11 PMID: 6246361
  32. Sequencing end-labeled DNA with base-specific chemical cleavages.
    Methods Enzymol. 1980;65(1):499-560 PMID: 6246368
  33. Biologial effects of the human complement fragments C5a and C5ades Arg on neutrophil function.
    Immunopharmacology. 1980 Jun;2(3):201-19 PMID: 6254906
  34. The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments.
    Nucleic Acids Res. 1980 Sep 11;8(17):3895-907 PMID: 7003539
  35. The expression in E. coli of synthetic repeating polymeric genes coding for poly(L-aspartyl-L-phenylalanine).
    Nucleic Acids Res. 1980 Oct 24;8(20):4575-92 PMID: 7003541
  36. Production of biologically active N alpha-desacetylthymosin alpha 1 in Escherichia coli through expression of a chemically synthesized gene.
    Biochemistry. 1980 Dec 23;19(26):6096-104 PMID: 7008828
  37. A system for shotgun DNA sequencing.
    Nucleic Acids Res. 1981 Jan 24;9(2):309-21 PMID: 6259625
  38. Response of human neutrophils to C5a: a role for the oligosaccharide moiety of human C5ades Arg-74 but not of C5a in biologic activity.
    J Immunol. 1981 Nov;127(5):1978-82 PMID: 7299121
  39. Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.
    Proc Natl Acad Sci U S A. 1981 Sep;78(9):5543-8 PMID: 6272309
  40. The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a.
    Crit Rev Immunol. 1981 Feb;1(4):321-66 PMID: 7037298
  41. Potentiation of the primary humoral immune response in vitro by C5a anaphylatoxin.
    J Immunol. 1982 Jul;129(1):70-5 PMID: 6177762
  42. A neutrophil chemotactic factor from human C'5.
    J Immunol. 1969 Jan;102(1):93-9 PMID: 5765461
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.82.11.3543
Published
1985-06-00
Pages
3543-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC397821
Subset
IM
Databases
GENBANK
K03392
External Links
PubMed source DOI: 10.1073/pnas.82.11.3543 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2