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 Escherichia coli strain. The soluble fusion protein of GST-sCT-Ala expressed from BL21(DE3)/pGEX-sCT-Ala was purified by affinity chromatography after high 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 precursor peptide was transacylated by CPD-Y, o-PNGA as a nucleophile, to produce photosensitive SO(-)(3)-sCT-o-PNGA. After photolysis and folding, the biological activity of sCT was assayed as standard.
Hong
D
D
Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, 200433, China.
Mingqiang
Z
Z
Min
L
L
Changqing
C
C
Jifang
M
M
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002116:Calcitonin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D006934:Hypercalcemia; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010782:Photolysis; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012474:Salmon
D007202:Indicators and Reagents; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002116:Calcitonin; D005982:Glutathione Transferase