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 collagenase-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 greater than or equal to 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.
Daum
J
J
Schering AG, PH-Biotechnologie, Berlin.
Donner
P
P
Geilen
W
W
Hübner-Kosney
G
G
Isernhagen
M
M
Scheidecker
H
H
Seliger
H
H
Boidol
W
W
Siewert
G
G
D000324:Adrenocorticotropic Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D003012:Microbial Collagenase / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D000324:Adrenocorticotropic Hormone; D004247:DNA; D000469:Alkaline Phosphatase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D003012:Microbial Collagenase