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Bulletin of Botanical Research ›› 2015, Vol. 35 ›› Issue (5): 724-729.doi: 10.7525/j.issn.1673-5102.2015.05.014

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Optimization of Prokaryotic Expression Factors for Blackberry UGT78H2 Using Responsive Surface Methodology and Protein Purification

CHEN Qing1;JIANG Lei-Yu1;WANG Yan1;ZHU Fang-Li1;TANG Hao-Ru1*;WANG Xiao-Rong1,2   

  1. 1.College of Horticulture,Sichuan Agricultural University,Ya’an 625014;
    2.Institute of Pomology and Olericulture,Sichuan Agricultural University,Chengdu 611130
  • Online:2015-09-15 Published:2015-11-20
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Abstract: 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-UGT78H2 plasmid was successfully transformed into the bacteria BL21(DE3)pLysS; (2)By RSM, incubating at 29.5℃ till cell OD600=0.51, IPTG was added to the final concentration of 0.4 mmol·L-1 to incubate for 7.4 hours, and the most quantities of recombinant UGT78H2 was obtained (166.4 μg·mL-1); (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.

Key words: blackberry, UGT78H2, prokaryotic expression, response surface methodology(RSM), recombinant protein

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