Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus
| Autor: | R C Anderson, William R. Mann, T M Johnson, Carol J. Dragland, Philip A. Bell, G M Nemecek |
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| Rok vydání: | 1995 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Spodoptera medicine.disease_cause Biochemistry law.invention Cell Line law Complementary DNA medicine Carnitine palmitoyltransferase II Animals Carnitine Cloning Molecular Molecular Biology Escherichia coli DNA Primers chemistry.chemical_classification biology Base Sequence Carnitine O-Palmitoyltransferase Cell Biology Molecular biology Recombinant Proteins Rats Isoenzymes Enzyme chemistry Liver Polyclonal antibodies biology.protein Recombinant DNA Specific activity Baculoviridae medicine.drug Research Article |
| Zdroj: | The Biochemical journal. 309 |
| ISSN: | 0264-6021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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