Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase
| Autor: | Linda A. Fothergill-Gilmore, Simon C. Allen, Teresa McNALLY, Hilary Muirhead, Toby H. L. Murcott |
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| Rok vydání: | 1991 |
| Předmět: |
Pyruvate dehydrogenase lipoamide kinase isozyme 1
Macromolecular Substances Protein Conformation Genetic Vectors Pyruvate Kinase Gene Expression Saccharomyces cerevisiae PKM2 Biology Pyruvate dehydrogenase phosphatase MAP3K7 Biochemistry Escherichia coli Promoter Regions Genetic Cyclin-dependent kinase 1 Circular Dichroism Phosphotransferases Cyclin-dependent kinase 3 Chromatography Ion Exchange Molecular biology Recombinant Proteins Kinetics Phosphotransferases (Alcohol Group Acceptor) Mutagenesis Chromatography Gel Cyclin-dependent kinase 7 Pyruvate kinase Plasmids |
| Zdroj: | European journal of biochemistry. 198(2) |
| ISSN: | 0014-2956 |
| Popis: | Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the covenient purification of 15–20 mg enzyme from 1 l culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described. |
| Databáze: | OpenAIRE |
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