Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.
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.
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D003182:Complement C5 / Q000235:genetics* / Q000494:pharmacology; D015936:Complement C5a; D004247:DNA / Q000138:chemical synthesis*; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D006168:Guinea Pigs; D006801:Humans; D008970:Molecular Weight; D009119:Muscle Contraction / Q000187:drug effects; D009841:Oligonucleotides / Q000138:chemical synthesis; D009876:Operon
D003182:Complement C5; D009841:Oligonucleotides; D015936:Complement C5a; D004247:DNA