Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme

被引:24
作者
Kawasaki, M
Makino, SI
Matsuzawa, H
Satow, Y
Ohya, Y
Anraku, Y
机构
[1] UNIV TOKYO,GRAD SCH SCI,DEPT BIOL SCI,BUNKYO KU,TOKYO 113,JAPAN
[2] UNIV TOKYO,FAC PHARMACEUT SCI,BUNKYO KU,TOKYO 113,JAPAN
[3] UNIV TOKYO,DEPT BIOTECHNOL,BUNKYO KU,TOKYO 113,JAPAN
关键词
D O I
10.1006/bbrc.1996.0826
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
VMA1 translational product undergoes excision of a 50-kDa intervening segment (VDE: VMA1-derived endonuclease) and religation of the flanking regions to create a 69-kDa catalytic subunit of vacuolar membrane H+-ATPase. VDEs conjugated with polypeptides at both N- and C-terminal ends were expressed in Escherichia coli and examined for their ability to catalyze self-splicing. Processed VDE was found in soluble pools, while unspliced precursors accumulated in insoluble pools, forming inclusion bodies. We demonstrate in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction efficiently occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed. (C) 1996 Academic Press, Inc.
引用
收藏
页码:827 / 832
页数:6
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