Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus.
The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively.
Johnson
T M
TM
Regulatory Toxicology Department, Sandoz Research Institute, Sandoz Pharmaceuticals Corp., East Hanover, New Jersey 07936-1080, USA.
Mann
W R
WR
Dragland
C J
CJ
Anderson
R C
RC
Nemecek
G M
GM
Bell
P A
PA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002334:Carnitine O-Palmitoyltransferase / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D002334:Carnitine O-Palmitoyltransferase