生物技术通报 ›› 2019, Vol. 35 ›› Issue (12): 10-15.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0475

• 研究报告 • 上一篇    下一篇

广藿香FPPS重组蛋白表达及互作蛋白筛选分析

钟李婷, 陈秀珍, 唐云, 李俊仁, 王小兵, 刘彦婷, 周璇璇, 詹若挺, 陈立凯   

  1. 广州中医药大学中药资源科学与工程研究中心 岭南中药资源教育部重点实验室(广州中医药大学)国家中成药工程技术研究中心南药研发实验室,广州 510006
  • 收稿日期:2019-05-30 出版日期:2019-12-26 发布日期:2019-12-03
  • 作者简介:钟李婷,女,硕士研究生,研究方向:植物遗传与调控;E-mail:13726857667@163.com
  • 基金资助:
    国家自然科学基金项目(81803657),广东省教育厅青年创新人才类项目(2017KQNCX039),广东省中医药局科技项目(20181075),广东省教育厅重点提升平台建设项目—岭南中药资源教育部重点实验室(2014KTSPT016)

Expression of FPPS Recombinant Protein from Pogostemon cablin and Screening of the Interaction Proteins

ZHONG Li-ting, CHEN Xiu-zhen, TANG Yun, LI Jun-ren, WANG Xiao-bing, LIU Yan-ting, ZHOU Xuan-xuan, ZHAN Ruo-ting, CHEN Li-kai   

  1. Research Center of Chinese Herbal Resource Science and Engineering,Guangzhou University of Chinese Medicine/Key Laboratory of Chinese Medicinal Resource from Lingnan(Guangzhou University of Chinese Medicine)/Ministry of Education,Guangzhou 510006
  • Received:2019-05-30 Published:2019-12-26 Online:2019-12-03

摘要: 对广藿香的法尼基焦磷酸酶(PatFPPS)进行重组蛋白的原核表达,并筛选与之互相作用的蛋白。利用PCR扩增PatFPPS编码区并连接到pGEX-6P-1表达载体,测序验证后将质粒转化BL21(DE3)表达菌株,并用IPTG诱导表达蛋白,最终得到带有GST标签的PatFPPS融合蛋白。利用GST Pull-Down技术,将GST-PatFPPS融合蛋白分别与广藿香叶片总蛋白进行体外孵育,洗脱得到蛋白复合物,经SDS-PAGE及LC-MS/MS鉴定。结果表明,PatFPPS原核表达体系成功建立,获得纯化重组蛋白,并且筛选得到一些候选PatFPPS互作蛋白。利用优化的原核表达体系,可获得可溶性的PatFPPS蛋白,并鉴定得到与之互作的候选蛋白。

关键词: 广藿香, 蛋白表达, 广藿香醇, 互作蛋白, 茉莉酸甲酯

Abstract: The purpose of this study is to express the recombinant protein of farnesyl pyrophosphatase synthase(PatFPPS)of Pogostemon cablin in prokaryotic cells and to screen its interaction proteins. The PatFPPS CDS was amplified and ligated into the pGEX-6P-1 vector by PCR. The plasmid confirmed by sequencing was obtained and transformed into BL21(DE3)expression strain,which was then induced by IPTG and the GST-tagged PatFPPS fusion protein was acquired. With GST Pull-Down technique,GST-tagged PatFPPS fusion protein was incubated with total proteins from the P. cablin leaves in vitro. The solution containing the protein complex was eluted and then identified by SDS-PAGE and LC-MS/MS. The results showed that the prokaryotic expression system of PatFPPS was successfully established and the purified recombinant protein was obtained,and some candidate interaction proteins were screened. In conclusion,soluble PatFPPS protein can be obtained with optimized prokaryotic expression system,and candidate proteins interacted with PatFPPS are identified.

Key words: Pogostemon cablin, protein expression, patchouli alcohol, interaction protein, methyl jasmonate