Expression, purification and biochemical comparison of natural and recombinant human non-pancreatic phospholipase A2.
The gene coding for human non-pancreatic phospholipase A2 (npPLA2) was cloned in a eukaryotic expression vector and transfected into chinese hamster ovary (CHO) cells. A number of cell lines stably expressing npPLA2 were obtained. Northern analysis of these cell lines showed an abundant transcript of expected size 1200 nt. The recombinant enzyme was efficiently secreted in quantities up to 400 micrograms npPLA2 per liter culture medium in the most productive cell lines. npPLA2 was purified to homogeneity from conditioned medium as previously described (1). The recombinant npPLA2 migrated by SDS--PAGE as a single band with an apparent mass of 14,000. The recombinant enzyme displayed the pH-optimum, calcium dependence and substrate preference that were characteristic of the human platelet and synovial fluid enzymes.
Johansen
B
B
Department of Botany/UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
Kramer
R M
RM
Hession
C
C
McGray
P
P
Pepinsky
R B
RB
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D015152:Blotting, Northern; D016466:CHO Cells / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010449:Peptide Mapping; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000201:enzymology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium