Production of human adrenocorticotropin by cleavage of alkaline‐phosphatase‐derived fusion proteins containing repetitive recognition sequences for collagenases

Recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to N‐terminal sequences of Escherichia coli alkaline phosphatase via collgenase‐sensitive linkers were constructed and used for the production of these proteins by transformed E. coli cells. It was shown that repetitive linkers of the form ‐Gly‐(Pro‐Xaa‐Gly)n‐Pro‐ with n≥2 were cleaved by clostridiopeptidase A (Clostridium histolyticum) by orders of magnitude faster than corresponding nonrepetitive sequences (n= 1). The C‐terminal cleavage product was Gly‐Pro‐adrenocorticotropin which could be converted to the authentic hormone by dipeptidyl peptidase IV. On the basis of these enzymatic reactions a procedure for the preparation of pure adrenocorticotropin was developed. Derivatives of alkaline phosphatase containing similar repetitive linker sequences were cleaved by clostridiopeptidase A as efficiently as the adrenocorticotropin fusion proteins.