%0 Journal Article
%A 2
%A CHEN Qing1
%A JIANG Lei-Yu1
%A TANG Hao-Ru1*
%A WANG Xiao-Rong1
%A WANG Yan1
%A ZHU Fang-Li1
%T Optimization of Prokaryotic Expression Factors for Blackberry UGT78H2 Using Responsive Surface Methodology and Protein Purification
%D 2015
%R 10.7525/j.issn.1673-5102.2015.05.014
%J Bulletin of Botanical Research
%P 724-729
%V 35
%N 5
%X UGT78H2 is a newly discovered glycosyltransferase from blackberry fruit, and the successfully obtained protein is the basis for further functional characterization. We constructed a prokaryotic expression vector and optimized the induction factors(induction temperature, cell and IPTG concentration, and induction duration) using responsive surface methodology(RSM). (1)the constructed pET32a-<em>UGT</em>78<em>H</em>2 plasmid was successfully transformed into the bacteria BL21(DE3)pLysS; (2)By RSM, incubating at 29.5℃ till cell OD<sub>600</sub>=0.51, IPTG was added to the final concentration of 0.4 mmol&middot;L<sup>-1</sup> to incubate for 7.4 hours, and the most quantities of recombinant UGT78H2 was obtained (166.4 &mu;g&middot;mL<sup>-1</sup>); (3)Incubation temperature and the length of culture time extremely significant affected the overall quantities of protein, and IPTG concentration and cell OD interactively determined the recombinant protein at significant level; (4)The recombinant protein was 67.9 kDa in molecular weight, existing as inclusion bodies in most fraction, and purified by utilizing Ni-NTA affiliation column system.
%U https://bbr.nefu.edu.cn/EN/10.7525/j.issn.1673-5102.2015.05.014