NUCLEOTIDE AND CORRECTED AMINO-ACID-SEQUENCE OF THE FUNCTIONAL RECOMBINANT RAT ANAPHYLATOXIN C5A

For bacterial expression of rat anaphylatoxin C5a, the cDNA was amplif ied by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat liver RNA and degenerate primers designed according to the publish ed amino acid sequence [1]. Surprisingly, the amino acid sequence dedu ced from cDNA differed at positions 55 (N for K), 63 (K for H), 67 (E for N), 68 (S for E) and 69 (H for S) from the published sequence. The overall amino acid composition, however, was unchanged because these 5 amino acids were located at different positions compared to the publ ished sequence. As a consequence, the proposed N-glycosylation site wa s absent, suggesting O-giycosylation of the mature molecule. Recombina nt rat C5a with a 6 histidine tag at the N-terminus was expressed in b acteria, purified and renatured. The peptide was as potent as recombin ant human C5a in eliciting lysosomal enzyme release from human granulo cytes. (C) 1997 Elsevier Science B.V.