[Studies on the expression, purification and renaturation of recombinant N-acety-L-ornithine deacetylase].
The argE gene from Escherichia coli coding for N-acety-L-ornithine deacetylase(NAOase), the key enzyme involved in the L-arginine biosynthesis, had been cloned in pET22b and transformed into BL21 (DE3). With 32.5% expression level in the optimal fermentation medium at 37 degrees C, most NAOase was expressed as inclusion bodies. The soluble and active proportion could be slightly increased when expressed at low temperature. The specific activity of soluble NAOase purified by Ni-NTA resin chromatography was 1193.2u/mg. The species and proportions of whole cell proteins varied with induction conditions. The inclusion bodies expressed at 37 degrees C was more pure than 22 degrees C after gradient wash with urea. Inclusion bodies could be partly refolding and reactivated by dilution and dialysis. Low protein concentration and suitable rate of oxidant/reducing agents were important to renaturation. In the optimal conditions 17.78% of Urea-denatured NAOase could be refolding and reactivated by dilution. The purified fusion protein was obtained after wash, solubilization and Ni-NTA resin affinity chromatography purification of inclusion bodies.
Li
Huan
H
College of Life Science and Pharmacy, Nanjing University of Technology, Nanjing 210009, China. lihuan45678@163.com
Chen
Yue
Y
Weng
Qiu-Ping
QP
Wu
Ming-Gang
MG
Wei
Ping
P
Ouyang
Ping-Kai
PK
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D002479:Inclusion Bodies / Q000201:enzymology; D017510:Protein Folding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014508:Urea; D000581:Amidohydrolases