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植物研究 ›› 2015, Vol. 35 ›› Issue (5): 724-729.doi: 10.7525/j.issn.1673-5102.2015.05.014

• 论文 • 上一篇    下一篇

黑莓果实UGT78H2重组蛋白诱导的响应面优化与蛋白纯化

陈清1;江雷雨1;王燕1;朱芳莉1;汤浩茹1*;王小蓉1,2   

  1. 1.四川农业大学园艺学院,雅安 625014;
    2.四川农业大学果蔬研究所,成都 611130
  • 出版日期:2015-09-15 发布日期:2015-11-20
  • 基金资助:
     

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
  • Supported by:
     

摘要: UGT78H2是从黑莓果实中新发现的一个植物糖基转移酶家族成员,获得重组蛋白是后续深入研究该基因功能的基础。本研究通过构建原核表达载体,并应用响应面分析方法对重组蛋白的诱导条件(如诱导温度、IPTG浓度、菌液浓度和诱导时间)进行了优化,结果表明:(1)构建了pET32a-UGT78H2原核表达载体,并成功导入到BL21(DE3)pLysS细菌中;(2)经响应面优化,在29.5℃培养工程菌至菌液OD600=0.51,加入终浓度为0.4 mmol·L-1的IPTG诱导7.4 h,可获得最大量重组蛋白166.4 μg·mL-1(占总蛋白41.6%);(3)诱导时间和培养温度极显著地影响重组蛋白表达量,诱导剂IPTG和诱导前菌液浓度之间的交互效应显著影响重组蛋白的表达;(4)获得的带S-tag和His-tag的UGT78H2重组蛋白分子量为67.9 kDa,主要以包涵体形式存在,通过Ni-NTA柱纯化,成功获得了重组蛋白。以上结果为进一步采用酶学方法进行UGT78H2蛋白功能鉴定提供了基础资料。

关键词: 黑莓, UGT78H2, 原核表达, 响应面分析, 重组蛋白

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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