食品科学 ›› 2017, Vol. 38 ›› Issue (2): 7-14.doi: 10.7506/spkx1002-6630-201702002

• 生物工程 • 上一篇    下一篇

小麦蛋白质二硫键异构酶基因的克隆、表达及重组酶性质

刘 光,胡松青,张婷婷,王敬敬,李 琳,侯 轶   

  1. 1.华南理工大学食品科学与工程学院,广东 广州 510640; 2.广东省天然产物绿色加工与产品安全重点实验室,广东 广州 510640
  • 出版日期:2017-01-25 发布日期:2017-01-16
  • 基金资助:
    国家自然科学基金面上项目(31471691;31130042);高等学校博士学科点专项科研基金项目(20130172110018);广东省科技计划项目(2014A010107002);佛山市科技计划项目(2015AG10011)

Gene Cloning, Expression and Characterization of Protein Disulfide Isomerase from Wheat (Triticum aestivum L.)

LIU Guang, HU Songqing, ZHANG Tingting, WANG Jingjing, LI Lin, HOU Yi   

  1. 1. School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China; 2. Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, Guangzhou 510640, China
  • Online:2017-01-25 Published:2017-01-16

摘要: 目的:克隆小麦蛋白质二硫键异构酶(wheat protein disulfide isomerase,wPDI)基因,实现其在大肠杆菌中的表达并探究其酶学性质。方法:以小麦种子总RNA为模板,逆转录并扩增得到wpdi,并以pET-30b为表达载体、大肠杆菌BL21(DE3)为宿主菌进行原核表达,表达产物经金属螯合层析纯化后进行了酶学性质研究。结果:克隆的基因全长1 548 bp,与“Wyuna”品种小麦wpdi基因相似性达99%。构建了pET-30b-wpdi表达载体,获得wPDI的最佳表达条件为:诱导温度22 ℃,诱导时间6 h,诱导剂浓度0.5 mmol/L。该酶含4 个硫氧还蛋白结构域,分子质量约为66.2 kD,具有二硫键的还原酶和异构酶活性以及分子伴侣活性。结论:对重组wPDI的表达和酶学性质研究,为wPDI在面制品加工及其他方面的应用提供参考依据。

关键词: 小麦蛋白质二硫键异构酶, 克隆, 表达, 酶学性质

Abstract: Purpose: The gene encoding wheat protein disulfide isomerase (wPDI) was cloned and expressed in Escherichiacoli, and its enzymatic properties were investigated. Methods: The wpdi gene was obtained by reverse transcriptionpolymerase chain reaction amplification using the total RNA from wheat seeds as template. The recombinant plasmid pET-30b-wpdi was constructed and transformed into E. coli BL21 (DE3). After metal chelating chromatography, the enzymaticproperties of the purified wPDI were determined. Results: A 1 548 bp gene fragment was amplified and sequenced as wpdigene that had 99% identity with that of the wheat cultivar Wyuna. The optimized conditions for wPDI expression weredetermined as follows: induction at 22 ℃ using isopropyl β-D-1-thiogalactopyranoside at a concentration of 0.5 mmol/L for6 h. The recombinant wPDI consisted of four thioredoxin-like domains and had a molecular weight of 66.2 kD. The enzymeexhibited enzymatic activities (including reductase activity and isomerase activity of disulfide bonds) and chaperone activity.Conclusions: The expression of wPDI and its enzymatic properties can provide the foundation for its application in the flourprocessing industry.

Key words: wheat protein disulfide isomerase, cloning, expression, enzymatic activities

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