Production in Escherichia coli of porcine type-I collagenase as a fusion protein with beta-galactosidase.
Porcine type-I collagenase (Colg-1) was produced as a fusion protein in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to Colg-1. Recombinant collagenase (reColg-1) was biologically active in the form of a fusion protein and could be released by treatment with factor Xa to yield Colg-1 with the authentic N terminus (phenylalanine) found in vivo. The results show that reColg-1 produced in E. coli is folded correctly, cleaves type-I collagen into 1/4 and 3/4 fragments at the characteristic Colg-sensitive site, and is produced at high enough levels to generate a source of recombinant enzyme for x-ray crystallography studies.
O'Hare
M C
MC
Rheumatology Research Unit, Addenbrooke's Hospital, Cambridge, U.K.
Clarke
N J
NJ
Cawston
T E
TE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003094:Collagen / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D003012:Microbial Collagenase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D003094:Collagen; D001616:beta-Galactosidase; D015951:Factor Xa; D003012:Microbial Collagenase