[Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris].
Human placental ribonuclease inhibitor is an acidic protein of Mr approximately 50 kDa with unusually high contents of leucine and cysteine. 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 paper, two molecules of alanine to substitute for cys328/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. The results indicated that there was no much change in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect was increased by 7-9 times. The enhance in anti-oxidative effect might be the reason for preventing the formation of disulfide bond between cys328 and cys329 and the three dimensional structure of RI was thereby maintained.
Wu
Yu
Y
College of Life Sciences, Liaoning Normal University, Dalian, China.
Zhao
Bao-Chang
BC
Wang
Ji-Hong
JH
Zhao
Peng
P
Wu
Yan-Ning
YN
Cui
Xiu-Yun
XY
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002384:Catalysis / Q000187:drug effects; D003545:Cysteine / Q000235:genetics; D018274:Electroporation; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010926:Placental Hormones / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism; D014170:Transformation, Genetic
D000975:Antioxidants; D004791:Enzyme Inhibitors; D010926:Placental Hormones; D011994:Recombinant Proteins; C022052:placental ribonuclease inhibitor; D006861:Hydrogen Peroxide; D012259:Ribonuclease, Pancreatic; D003545:Cysteine; D000409:Alanine