Expression of the Trypanosoma brucei phosphoenolpyruvate carboxykinase gene in Saccharomyces cerevisiae

被引：3

作者：

Yévenes, A ^{[1
]}

Cardemil, E ^{[1
]}

机构：

[1] Univ Santiago Chile, Fac Quim & Biol, Dept Ciencias Quim, Santiago 33, Chile

来源：

BIOCHIMIE | 2000年 / 82卷 / 02期

关键词：

phosphoenolpyruvate carboxykinase Trypanosoma brucei; Saccharomyces cerevisiae; gene expression;

D O I：

10.1016/S0300-9084(00)00189-9

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Plasmid pTbp60B (Kueng et al., J. Biol. Chem. 264 (1989) 5203-5209) was employed to obtain, through the polymerase chain reaction, the Trypanosoma brucei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then cloned into the yeast expression plasmid pYES2. The cloned gene was completely sequenced and the expression plasmid transformed into Saccharomyces cerevisiae PUK-3B (MAT alpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant I: brucei PEP carboxykinase was purified to near homogeneity. The basic molecular and catalytic characteristics of the recombinant enzyme were determined, and they showed to be essentially similar to those reported for wild type I: brucei PEP carboxykinase (Hunt and Kohler, Biochim. Biophys. Acta 1249 (1995) 15-22). The expression system here described is a reliable non-pathogenic source of T. brucei PEP carboxykinase. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS.

引用

页码：123 / 127

页数：5

共 19 条

[1]

Bryant C., 1993, P117, DOI 10.1002/9781444313963.ch5

[2]

CARDEMIL E, 1999, PROTEIN SCI, V8, pR40

[3] AEROBIC FERMENTATION OF GLUCOSE BY TRYPANOSOMATIDS [J].

CAZZULO, JJ .

FASEB JOURNAL, 1992, 6 (13) :3153-3161

[4] Methodology for the preparation of pure recombinant S-cerevisiae lanosterol synthase using a Baculovirus expression system. Evidence that oxirane cleavage and A-ring formation are concerted in the biosynthesis of lanosterol from 2,3-oxidosqualene [J].

Corey, EJ ;

Cheng, HM ;

Baker, CH ;

Matsuda, SPT ;

Li, D ;

Song, XL .

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1997, 119 (06) :1277-1288

[5]

Cox F. E. G., 1993, P1, DOI 10.1002/9781444313963.ch1

[6]

Han YM, 1999, APPL ENVIRON MICROB, V65, P1915

[7] PURIFICATION AND CHARACTERIZATION OF PHOSPHO ENOLPYRUVATE CARBOXYKINASE FROM TRYPANOSOMA-BRUCEI [J].

HUNT, M ;

KOHLER, P .

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1995, 1249 (01) :15-22

[8] The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer [J].

Krautwurst, H ;

Bazaes, S ;

González, FD ;

Jabalquinto, AM ;

Frey, PA ;

Cardemil, E .

BIOCHEMISTRY, 1998, 37 (18) :6295-6302

[9]

KUENG V, 1989, J BIOL CHEM, V264, P5203

[10] Cloning, sequencing, and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene [J].

Laivenieks, M ;

Vieille, C ;

Zeikus, JG .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63 (06) :2273-2280

← 1 2 →