生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (3): 47-.doi: 10.3969/j.issn.2095-1736.2021.03.047

• 研究报告 • 上一篇    下一篇

毕赤酵母GT1的原核表达与体外功能鉴定

  

  1. 1.江南大学 粮食发酵工艺与技术国家工程实验室，无锡214122；
    2. 江南大学 工业生物技术教育部重点实验室，无锡214122；
    3. 江南大学 生物工程学院糖化学与生物技术教育部重点实验室，无锡214122
  • 出版日期:2021-06-18 发布日期:2021-06-21
  • 通讯作者: 杨艳坤，副教授，硕士生导师，主要从事微生物学、分子生物学、发酵工程研究，E-mail：yangyankun@jiangnan.edu.cn
  • 作者简介:潘颖越，硕士，主要从事发酵工程、分子生物学研究，E-mail：panyingyuejndx@163.com
  • 基金资助:
    国家自然科学基金项目(31570034)； 江苏省第十五批六大人才高峰项目(SWYY-180)

Prokaryotic expression and function identification of Pichia pastoris GT1 in vitro #br#

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology, Wuxi 214122, China;
    2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Wuxi 214122, China;
    3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology,
    Jiangnan University, Wuxi 214122, China
  • Online:2021-06-18 Published:2021-06-21

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摘要： 为了开展体外互作实验，以pXMJ19质粒为载体构建pXMJ19-gt1-His重组质粒，通过单因素优化实验获得毕赤酵母甘油转运体GT1的最优表达宿主为C41(DE3)；最优表达条件为OD600达到0.7时加入终浓度为0.1mmol/L的异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)，于30℃、110r/min诱导10h。通过超速离心验证了部分GT1蛋白在大肠杆菌中位于细胞质膜上，并筛选得到针对GT1重溶解率最高的去垢剂NP-40。在去垢剂环境下通过镍离子亲和层析纯化得到GT1蛋白，然后利用等温滴定量热法体外检测其与甘油的结合能力，结果表明:GT1与甘油在体外相互作用产生放热反应，且测得其与甘油的平衡解离常数KD值为6.58μmol/L。实验说明原核表达的GT1蛋白具有活性，且与甘油的体外相互作用明显，为GT1以甘油为配体进行结晶以及解析其三维结构提供了研究基础。

关键词: 巴斯德毕赤酵母, 甘油转运体, 膜蛋白表达, MFS蛋白超家族, 蛋白-分子相互作用

Abstract: In order to perform isothermal titration calorimetry (ITC) experimentsin vitro, pXMJ19-gt1-His recombinant plasmid was constructed.Selected by single-factor optimization experiments, the optimal host cell for the glycerol transporterGT1expression was C41(DE3); the optimal expression condition was adding 0.01mmol/L Isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 0.7 and inducing at 30℃, 110r/min for 10h. It was identified by ultracentrifugation that some GT1 protein could be located to the plasma membrane of Escherichia coli. The detergent NP-40 with the highest resolving efficiency for GT1 was screened out. The GT1 protein was purified using nickel ion affinity chromatography with detergent buffer, and its glycerol binding affinity was measured by ITC. The results showed that the binding process was exothermic reaction, GT1 interacted with glycerol in vitro with a KDvalue of 6.58μmol/ L. The experiments showed that the GT1 protein was active and it could interact with glycerol in vitro, which provided a research basis for the study on crystal structureand function of GT1.

Key words: Pichia pastoris, glycerol transporter, membrane protein expression, major facilitator superfamily, protein-molecule interaction

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