Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast

被引:28
作者
Lanfermeijer, FC
Venema, K
Palmgren, MG
机构
[1] Univ Copenhagen, Inst Mol Biol, DK-1353 Copenhagen K, Denmark
[2] Royal Vet & Agr Univ, Dept Plant Biol, DK-1871 Frederiksberg, Denmark
关键词
D O I
10.1006/prep.1997.0788
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
In order to facilitate efficient purification of the plant plasma membrane H+-ATPase expressed in yeast, a recombinant H+-ATPase protein with an N-terminal affinity tag of six histidine residues was engineered, When expressed in yeast the recombinant protein accumulated in the endoplasmic reticulum in an active form and showed characteristics comparable with those of the wildtype plasma membrane H+-ATPase (K-m,K-ATP, 1.1 mM; pH optimum, 6.6), After solubilization of the membrane proteins from the endoplasmic reticulum with n-dodecyl-beta-D-maltoside, the recombinant protein was purified under nondenaturing conditions by chromatography on Ni2+-nitriloacetic acid-agarose. A fraction was obtained which contained 4.2% of the initial amount of the protein and 26.6% of the ATPase-activity present in the endoplasmic reticulum, The purified protein has a specific activity of 32.6 mu mol min(-1) mg protein(-1) at pH 6.5 and 30 degrees C, This rate is equivalent to a molecular activity of 3400 min(-1), The purified plasma membrane H+-ATPase could be reconstituted into liposomes and demonstrated in this configuration the ability to pump protons. The method proves to be a convenient and rapid method for the preparation of purified single isoforms and mutant proteins of the plant plasma membrane H+-ATPase in high and functional quantities, This method might also be useful for achieving purification of other P-type ATPases, normally expressed at very low levels in heterologous systems. (C) 1998 Academic Press.
引用
收藏
页码:29 / 37
页数:9
相关论文
共 41 条
[1]  
ADAMO HP, 1988, J BIOL CHEM, V263, P17548
[2]   PURIFICATION AND PROPERTIES OF THE H+-TRANSLOCATING ATPASE FROM THE PLASMA-MEMBRANE OF TOMATO ROOTS [J].
ANTHON, GE ;
SPANSWICK, RM .
PLANT PHYSIOLOGY, 1986, 81 (04) :1080-1085
[3]   DETERMINATION OF PHOSPHATE - STUDY OF LABILE ORGANIC PHOSPHATE INTERFERENCE [J].
BAGINSKI, ES ;
FOA, PP ;
ZAK, B .
CLINICA CHIMICA ACTA, 1967, 15 (01) :155-&
[4]   AMINO-ACID ANALYSIS - DETERMINATION OF CYSTEINE PLUS HALF-CYSTINE IN PROTEINS AFTER HYDROCHLORIC-ACID HYDROLYSIS WITH A DISULFIDE COMPOUND AS ADDITIVE [J].
BARKHOLT, V ;
JENSEN, AL .
ANALYTICAL BIOCHEMISTRY, 1989, 177 (02) :318-322
[5]  
BEGUIN P, 1993, J BIOL CHEM, V268, P15958
[6]  
BLAKE MS, 1984, ANAL BIOCHEM, V128, P302
[7]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[8]   SUPERPOLYLINKERS IN CLONING AND EXPRESSION VECTORS [J].
BROSIUS, J .
DNA-A JOURNAL OF MOLECULAR & CELLULAR BIOLOGY, 1989, 8 (10) :759-777
[9]   A YEAST GENE THAT IS ESSENTIAL FOR RELEASE FROM GLUCOSE REPRESSION ENCODES A PROTEIN-KINASE [J].
CELENZA, JL ;
CARLSON, M .
SCIENCE, 1986, 233 (4769) :1175-1180
[10]   REPLACEMENT OF THE PROMOTER OF THE YEAST PLASMA-MEMBRANE ATPASE GENE BY A GALACTOSE-DEPENDENT PROMOTER AND ITS PHYSIOLOGICAL CONSEQUENCES [J].
CID, A ;
PERONA, R ;
SERRANO, R .
CURRENT GENETICS, 1987, 12 (02) :105-110