黑莓果实UGT78H2重组蛋白诱导的响应面优化与蛋白纯化

doi:10.7525/j.issn.1673-5102.2015.05.014

摘要/Abstract

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

中图分类号:

陈清1；江雷雨1；王燕1；朱芳莉1；汤浩茹1*；王小蓉1,2. 黑莓果实UGT78H2重组蛋白诱导的响应面优化与蛋白纯化[J]. 植物研究, 2015, 35(5): 724-729.

CHEN Qing1；JIANG Lei-Yu1；WANG Yan1；ZHU Fang-Li1；TANG Hao-Ru1*；WANG Xiao-Rong1,2. Optimization of Prokaryotic Expression Factors for Blackberry UGT78H2 Using Responsive Surface Methodology and Protein Purification[J]. Bulletin of Botanical Research, 2015, 35(5): 724-729.

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