Production of recombinant salmon calcitonin by amidation of precursor peptide using enzymatic transacylation and photolysis in vitro

The C terminal amidation is required for full biological activity of salmon calcitonin (sCT), We constructed BL21(DE3)/pGEX-sCT-Ala, an engineering Es cherichia coli strain. The soluble fusion protein of GST-sCT-Ala expressed from BL21(DE3)/pGEX-sCT-Ala was purified by affinity chromatography after h igh density, high expression culture and sonication of bacteria. Following S-sulfonation of the fusion protein, the 33 alanine-extended peptides were released from the fusion protein by cyanogen bromide. The S-sulfonated prec ursor peptide was transacylated by CPD-Y, o-PNGA as a nucleophile, to produ ce photosensitive SO3--sCT-o-PNGA. After photolysis and folding, the biolog ical activity of sCT was assayed as standard. (C) 2000 Academic Press.