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

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Abstract
A gene coding for the C5a fragment of the 5th component of human complement was chemically synthesized, cloned and expressed in E. coli. The 253-base-pair gene fragment was built through a 2-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 uppromoter 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 feasibility of expressing C5a anaphylatoxin in bacteria is demonstrated and a system for mutagenesis of the C5a protein is provided.