EXPORT AND PURIFICATION OF A CYTOPLASMIC DIMERIC PROTEIN BY FUSION TO THE MALTOSE-BINDING PROTEIN OF ESCHERICHIA-COLI

被引:20
作者
BLONDEL, A [1 ]
BEDOUELLE, H [1 ]
机构
[1] INST PASTEUR, UNITE BIOCHIM CELLULAIRE,CNRS,UNITE RECH D1129, 28 RUE DOCTEUR ROUX, F-75724 PARIS 15, FRANCE
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1990年 / 193卷 / 02期
关键词
D O I
10.1111/j.1432-1033.1990.tb19341.x
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
A hybrid between the maltose‐binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE‐G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE‐G5P was purified from a periplasmic extract by affinity chromatography on cross‐linked amylose, with a yield larger than 50000 molecules/E. coli cell. The hybrid specifically bound denatured but not double‐stranded DNA cellulose, as native G5P. Sedimentation velocity and gel‐filtration experiments showed that MalE‐G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE‐G5P will enable the study of mutant G5P that no longer binds single‐stranded DNA and therefore cannot be purified by DNA‐cellulose chromatography. Copyright © 1990, Wiley Blackwell. All rights reserved
引用
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页码:325 / 330
页数:6
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