SYRIAN-HAMSTER 3-HYDROXY-3-METHYLGLUTARYL-COENZYME-A REDUCTASE EXPRESSED IN ESCHERICHIA-COLI - PRODUCTION OF HOMOGENEOUS PROTEIN

被引:28
作者
FRIMPONG, K
DARNAY, BG
RODWELL, VW
机构
[1] Department of Biochemistry, Furdue Unversity, West Lafayette
[2] University of Texas, M. D. Anderson Cancer Center, Cytokine Research Section, Houston, TX 77030-4095, CIBT-041
关键词
D O I
10.1006/prep.1993.1044
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 over-expression, 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 μmol 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. © 1993 Academic Press. All rights reserved.
引用
收藏
页码:337 / 344
页数:8
相关论文
共 28 条
[1]   CLONING, SEQUENCING, AND OVEREXPRESSION OF MVAA, WHICH ENCODES PSEUDOMONAS-MEVALONII 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE [J].
BEACH, MJ ;
RODWELL, VW .
JOURNAL OF BACTERIOLOGY, 1989, 171 (06) :2994-3001
[2]   BIOSYNTHESIS AND CHARACTERIZATION OF (S) AND (R)-3-HYDROXY-3-METHYLGLUTARYL COENZYME-A [J].
BISCHOFF, KM ;
RODWELL, VW .
BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY, 1992, 48 (02) :149-158
[3]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[4]  
DARNAY BG, 1992, J BIOL CHEM, V267, P15064
[5]  
EDWARDS PA, 1980, J BIOL CHEM, V255, P3715
[6]  
ENDO A, 1992, J LIPID RES, V33, P1569
[7]   EXPRESSION OF CATALYTICALLY ACTIVE RADISH 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE IN ESCHERICHIA-COLI [J].
FERRER, A ;
APARICIO, C ;
NOGUES, N ;
WETTSTEIN, A ;
BACH, TJ ;
BORONAT, A .
FEBS LETTERS, 1990, 266 (1-2) :67-71
[8]   REGULATION OF THE MEVALONATE PATHWAY [J].
GOLDSTEIN, JL ;
BROWN, MS .
NATURE, 1990, 343 (6257) :425-430
[9]   EUKARYOTIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI - AN IMPROVED THROMBIN CLEAVAGE AND PURIFICATION PROCEDURE OF FUSION PROTEINS WITH GLUTATHIONE-S-TRANSFERASE [J].
GUAN, KL ;
DIXON, JE .
ANALYTICAL BIOCHEMISTRY, 1991, 192 (02) :262-267
[10]   A FAST AND SIMPLE PROCEDURE FOR SEQUENCING DOUBLE-STRANDED DNA WITH SEQUENASE [J].
HSIAO, KC .
NUCLEIC ACIDS RESEARCH, 1991, 19 (10) :2787-2787