Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli.
Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.
Seddi
Rachid
R
Institut Méditerranéen de Recherche en Nutrition, Service 342, UMR Université Aix-Marseille III-INRA 1111, Faculté des Sciences et Techniques de Saint-Jérôme, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France.
Chaix
Jean-Claude
JC
Puigserver
Antoine
A
Guo
Xiao-Jun
XJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism; D043422:Carboxypeptidases A; D002417:Cattle; D002845:Chromatography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D014357:Trypsin