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Frontiers in Biology : Volume 1, Issue 1, January 2006

Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris

Content Provider	Springer Nature Link
Author	Wu, Yu Cui, Xiuyun Wang, Jihong Zhao, Peng Xu, Yuefei Zhao, Baochang
Copyright Year	2006
Abstract	Human placental ribonuclease inhibitor (hRI) is an acidic protein of Mr∼50kDa with unusually high contents of leucine and cysteine residues. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. hRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence. In the present aork, two molecules of alanine substituting for Cys328 and Cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K and then transformed Pichia pastoris GS115 by electroporation. After colony screening, the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. Results indicated that there were not many changes in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect increased by 7∼9 times. The enhancement in anti-oxidative effect might be attributed to preventing the formation of disulfide bond between Cys328 and Cys329 and the three dimensional structure of RI was thereby maintained.
Starting Page	99
Ending Page	103
Page Count	5
File Format	PDF
ISSN	16733509
Journal	Frontiers in Biology
Volume Number	1
Issue Number	2
e-ISSN	16733622
Language	English
Publisher	Higher Education Press
Publisher Date	2006-01-01
Publisher Institution	Chinese Universities
Publisher Place	Beijing
Access Restriction	Subscribed
Subject Keyword	ribonuclease inhibitor site-directed mutagenesis cysteine Pichia pastoris reactive oxygen species Life Sciences
Content Type	Text
Resource Type	Article
Subject	Ecology Genetics Ecology, Evolution, Behavior and Systematics Biotechnology

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