Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase expressed in Escherichia coli: production of homogeneous protein.
When overexpressed in Escherichia coli, the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) is catalytically active, but exhibits major heterogeneity. This heterogeneity reflects deletion of about 60 aminoacyl residues from the C-terminus, presumably a result of proteolytic cleavage or premature termination of translation. With the intent of separating the intact and truncated proteins via immunoaffinity chromatography, we constructed the expression phagemid pKFT7-21. This construct encodes the catalytic domain of Syrian hamster HMG-CoA reductase with the C-terminal extension Glu-Glu-Phe, an epitope recognized by a specific antibody. Following overexpression, the modified catalytic domain RcatEEF had high catalytic activity and exhibited no heterogeneity. It therefore was possible to purify RcatEEF to over 95% homogeneity without resorting to immunoaffinity chromatography. The yield of homogeneous protein averaged 20-25 mg per liter of cells with a final specific activity of up to 40 mumol NADPH oxidized per minute per milligram. The EEF modification thus should prove useful for the purification of the catalytic domains of other eukaryotic HMG-CoA reductases which exhibit heterogeneity.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
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Darnay
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D005982:Glutathione Transferase