Purification of pancreatic cholesterol esterase expressed in recombinant baculovirus-infected Sf9 cells.
A cDNA clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins (M(r) 74 kDa and 64 kDa) reactive with anti-cholesterol esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 micrograms/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of cholesterol esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 micrograms/ml at Day 8. Both the 74- and the 64-kDa cholesterol esterases were biologically active and hydrolyzed the artificial substrate p-nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic cholesterol esterase. The recombinant enzyme will be useful for further characterization of this protein.
DiPersio
L P
LP
Department of Pathology, University of Cincinnati College of Medicine, Ohio 45267-0529.
Kissel
J A
JA
Hui
D Y
DY
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D009036:Moths; D010179:Pancreas / Q000201:enzymology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives; D002787:Sterol Esterase / Q000096:biosynthesis / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; C037405:heparin-sepharose; D004247:DNA; D012685:Sepharose; D002787:Sterol Esterase