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

Abstract

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia cofl. 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 uppromoter mutant, UVS-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a2purified from human serum in inhibiting binding of human 1 I-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.Activation of the complement system generates several biologically active peptides. One of these, C5a, is a potent stimulant of inflammatory processes and potentially plays an important role in the pathogenesis of a number of inflammatory diseases (1, 2). C5a is a glycoprotein ofMr 11,000 (3). During serum complement activation, CSa is cleaved from the amino end of the a chain of the fifth component of complement, C5, by a trypsin-like enzyme, C5 convertase (4). Once CSa is liberated, the carboxyl-terminal arginine is rapidly removed by serum carboxypeptidase N, forming the desarginine derivative, des-Arg-C5a (5). CSa is a potent stimulator of a variety of neutrophil functions thought to be involved in inflammation, including chemotaxis, chemokinesis, aggregation, lysosomal enzyme release, and the generation of toxic oxygen products (6-10). In addition, C5a produces smooth muscle contraction and vasodilation (11,12), promotes release of histamine from basophils and mast cells (13,14), and stimulates antibody synthesis (15). Des-Arg-C5a is capable of producing many of these effects but is less potent than C5a (7,12).Although the sequence of its 74 amino acids has been elucidated (3), efforts to determine the secondary and tertiary structure of C5a and to relate these features to the function of the molecule have been limited by difficulties in obtaining suitable quantities of C5a. In a typical isolation, <0.5 mg of pure C5a can be recovered from 1 liter of complement-activated serum (16). For this reason, there is a paucity of sequence-homology information, as only one other mammalian C5a (porcine) has been sequenced (17). The ability to test additional variant or mutant forms of C5a would provide insight concerning which residues are important for its biological activity. Recent advances in recombinant DNA technology now make it feasible both to approach the problem of inadequate supply of a scarce protein and to generate a system for mutagenesis by clonin...

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

“…Mutant C5a-encoding genes were constructed by either replacing a restriction fragment of the native CSa-encoding gene (16) by the double-stranded synthetic DNA that carried the mutated sequence or by oligonucleotide-directed double-strand break repair (17). Expression vector, host strain (18), and fermentation conditions (19) were as previously described.…”

Elastase Controls the Binding of the Vitamin D-Binding Protein (Gc-Globulin) to Neutrophils: A Potential Role in the Regulation of C5a Co-Chemotactic Activity

“…Human rC5a was a generous gift from Karl Mollison of Abbott Laboratories (Abbott Park, IL), and was prepared using an Escherichia coli expression system, as previously described (38). Purified human DBP was purchased from Biodesign International (Kennebunkport, ME).…”

The pharmacophore of the human C5a anaphylatoxin

“…A synthetic gene (based on Mandecki et al, 1985) that encodes the 74-amino acid human C5a protein was used as the wild-type allele (Armenti, 1989). This C5a gene was assembled from several oligonucleotides and cloned into M13mp18 to give M13mplWC5a.…”

“…However, our recombinant C5a was found to be equally potent to authentic human serum C5a in binding affinity and in CSa-induced intracellular calcium rise (data not shown). A similar synthetic C5a gene was expressed and purified from E. coli in 2 other laboratories (Mandecki et al, 1985;Franke et al, 1988). The recombinant C5a from both of these groups contained a methionine at the N-terminus before the initial threonine residue of human C5a.…”