HIGH-LEVEL SYNTHESIS IN ESCHERICHIA-COLI OF RECOMBINANT HUMAN CALCITONIN - COLLAGENASE CLEAVAGE OF THE FUSION PROTEIN AND PEPTIDYLGLYCINE ALPHA-AMIDATION

A synthetic gene encoding glycine-extended human calcitonin (hCT-GlY) was inserted between the tac-promoter and the lacZ gene. A synthetic DNA with collagenase recognition sites was placed just downstream at the hCT-Gly gene. In induced cultures, the E. coli cells harboring the gene produced about 0.8 g of the fusion protein (22 mg of hCT-Gly) per liter of culture. The fusion protein was easily and precisely cleaved with a bacterial collagenase to obtain the hCT-Gly. The hCT-Gly was converted with a peptidylglycine alpha-amidating enzyme to mature hCT, which had hypocalcemic activity similar to that of authentic hCT. These results demonstrate that expression of the fusion gene containing collagenase recognition sites is an effective method for the high-level production of intact peptide hormones.