Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme.
The serum activity of human pancreatic lipase (HPL; EC 3.1.1.3), the main lipolytic enzyme secreted by the pancreas, is a valuable marker of pancreatic disorders. However, determining lipase activity in human serum is difficult because the substrates used vary in their lipase specificity. A lipase reference material is therefore needed. We describe the production of recombinant HPL (rHPL) in V79 Chinese hamster lung cells. A cDNA encoding the sequence of HPL was subcloned into the pcDNAI eukaryotic expression vector, which was then used to transfect V79 cells. The 50-kDa purified recombinant enzyme is fully active, is glycosylated, and has a pI of 7.0. The catalytic properties of rHPL, determined by using triolein as the substrate, were similar to those of the native enzyme. The V79rHPL cell line we developed might be useful for the production of rHPL suitable for the preparation of a reference material.
Centre du Médicament, URA CNRS 597 30, Nancy, France.
F
F
Canalias
A
A
Visvikis
C
C
Thioudellet
G
G
Siest
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D007526:Isoelectric Point; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008168:Lung; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D003470:Culture Media; D018076:DNA, Complementary; D011994:Recombinant Proteins; D008049:Lipase