[49] Human C5a anaphylatoxin: Gene synthesis, expression, and recovery of biologically active material from Escherichia coli

Anaphylatoxin C5a is a 74-amino acid residue single-chain glycoprotein derived from the fifth component of complement during the activation of the complement cascade. As quantities of C5a, which can be purified from serum, especially human, are extremely limited, many extensive biological and biochemical/biophysical investigations are made difficult. The production of biologically active C5a by recombinant DNA techniques would provide adequate quantities of anaphylatoxin for such studies and also provide the opportunity to examine its protein structure-activity relationships by site-specific mutagenesis. The plan devised to achieve bacterial production of human anaphylatoxin C5a is analogous to previous methods used for the synthesis and expression of several small human hormones. It employs the construction of a gene in which the coding sequence for the activated complement component C5a is made by chemical synthesis. To produce anaphylatoxin in E. coli, it is necessary to place a bacterial promoter and ribosome-binding site sequence upstream of the C5a gene. The construction of the C5a expression plasmid is initiated by the cleavage of the C5a subclone pBR322-C5a with the restriction endonudease EcoRI followed by dephosphorylation using bacterial alkaline phosphatase (BAP).