OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION AND CHARACTERIZATION OF THE MOLECULAR CHAPERONE HSC70

被引:20
作者
BENAROUDJ, N
FANG, B
TRINIOLLES, F
GHELIS, C
LADJIMI, MM
机构
[1] CNRS,ENZYMOL LAB,F-91198 GIF SUR YVETTE,FRANCE
[2] UNIV PARIS 11,BIOL PHYSIOCHIM LAB,ORSAY,FRANCE
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1994年 / 221卷 / 01期
关键词
D O I
10.1111/j.1432-1033.1994.tb18720.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
The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 1l of cell culture. Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.
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页码:121 / 128
页数:8
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