Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme.
Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity. Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein. We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding. While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation. This treatment improves the recovery of active CK approximately 100-fold. We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E. coli. Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well.
Babbitt
P C
PC
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
West
B L
BL
Buechter
D D
DD
Kuntz
I D
ID
Kenyon
G L
GL
D000818:Animals; D007611:Aprotinin / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005960:Glucosides / Q000494:pharmacology; D002479:Inclusion Bodies; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D014101:Torpedo
D005960:Glucosides; C018619:octyl-beta-D-glucoside; D007611:Aprotinin; D003402:Creatine Kinase; D010447:Peptide Hydrolases