MELIBIOSE PERMEASE OF ESCHERICHIA-COLI - LARGE-SCALE PURIFICATION AND EVIDENCE THAT H+, NA+, AND LI+ SUGAR SYMPORT IS CATALYZED BY A SINGLE POLYPEPTIDE

被引:95
作者
POURCHER, T
LECLERCQ, S
BRANDOLIN, G
LEBLANC, G
机构
[1] CEA,DEPT BIOL CELLULAIRE & MOLEC,LAB JEAN MAETZ,F-06230 VILLEFRANCHE MER,FRANCE
[2] CEN GRENOBLE,CEA,DEPT BIOL MOLEC & STRUCT,BIOCHIM LAB,CNRS,UA 1130,F-38054 GRENOBLE 9,FRANCE
关键词
D O I
10.1021/bi00013a033
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
As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin, After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence, Purified permeases, reconstituted in liposomes, display H+-, Na+-, or Li+-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide.
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页码:4412 / 4420
页数:9
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