Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.
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 convenient purification of 15-20 mg enzyme from 11 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.
Murcott
T H
TH
Department of Biochemistry, School of Medical Sciences, University of Bristol, England.
McNally
T
T
Allen
S C
SC
Fothergill-Gilmore
L A
LA
Muirhead
H
H
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D016296:Mutagenesis; D010770:Phosphotransferases / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020670:glycerate kinase; D011770:Pyruvate Kinase