芽孢杆菌β-折叠桶植酸酶的原核可溶性表达优化及包涵体复性研究

中国生物工程杂志

2012, Vol. 32

Issue (08): 49-55

研究报告

芽孢杆菌β-折叠桶植酸酶的原核可溶性表达优化及包涵体复性研究

李倩倩¹, 李中媛², 冯舵¹, 黄火清², 韩翠晓¹, 杨培龙², 姚斌², 高伟¹

1. 北京林业大学理学院/林木育种国家工程实验室北京 100083;
2. 中国农业科学院饲料研究所农业部饲料生物技术重点实验室北京 100081

Optimizing Soluble Expression and Inclusion Body Renature Research of β-Propeller Phytase of Bacillus sp. HJB17 in E.coli

LI Qian-qian¹, LI Zhong-yuan², FENG Duo¹, HUANG Huo-qing², HAN Cui-xiao¹, YANG Pei-long², YAO Bin², GAO Wei ¹

1. National Engineering Laboratory for Tree Breeding /College of Science,Beijing Forestry University,Beijing 100083,China;
2. Key Laboratory for Feed Biotechnology of the Ministry of Agriculture,Feed Research Institute,Chinese Academy of Agricultural Sciences, Beijing 100081,China

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摘要： 目的:来源于芽孢杆菌的β-折叠桶植酸酶基因PhyH,截去N端120个碱基编码的40个氨基酸后,成功构建了原核表达体系,通过两种方法分别得到有活性的目的蛋白PhyHT,并通过进一步纯化提高目的蛋白的纯度。方法:通过分子伴侣共表达系统提高目的蛋白的可溶性表达,并通过包涵体复性研究,从包涵体中制备出有活性的目的蛋白。结果:(1)目的蛋白PhyHT主要以包涵体形式存在于沉淀中;(2)通过优化表达条件,降低温度和诱导剂浓度均不能明显改善包涵体问题,通过构建分子伴侣共表达系统(即pG-KJE8、pGro7、pKJE7和 pTf16 4种分子伴侣质粒分别与重组表达质粒pET28b-PhyHT共表达),筛选能提高目的蛋白可溶性表达的分子伴侣质粒;(3)包涵体经过复性和进一步的纯化,得到了高纯度的有生物活性的目的蛋白。

关键词： β-折叠桶植酸酶PhyHT; 分子伴侣; 可溶性表达; 包涵体; 蛋白纯化

Abstract: PhyH,a novel phytase from Bacillus sp. HJB17,successfully clone and express the truncated phytase PhyHT(cancel the first 120 nucleotides which encode a 40 amino acid secreted signal peptide) in Escherichia coli. To improve the soluble expression level of PhyHT, and get large-scale purified and active proteins,the expression conditions were optimized,including concentrations of IPTG and temperatures of induction. The co-expression system of pET28b-PhyHT and four molecular chaperone vectors(pG-KJE8,pGro7,pKJE7, and pTf16)were constructed respectively.Additionally,it was successful in vitro refolding of Bacillus phytase from the inclusion bodies. The results were described as follows: (1) the PhyHT gene was cloned into vector pET28b successfully ; the expressed proteins were found in the insoluble cytoplasmic fraction as inclusion bodies, even with different expression conditions.(2) Molecular chaperone vectors pGro7 and pKJE7 improved significantly the soluble protein level.(3)PhyHT with biological activity was obtained after the recombinant vector pET28b-PhyHT was expressed and renatured. Furthermore,the protein was purified through Ni-NTA and gel filtration chromatography. The soluble proteins provide a potential value for the further mechanistic study of the structure and function of PhyHT.

Key words: β-Propeller phytase PhyHT Soluble expression Molecular chaperone Inclusion body Protein purification

收稿日期: 2012-04-27 出版日期: 2012-08-25

ZTFLH:

Q819

基金资助: 国家自然科学基金(30970578 31070651)、教育部新世纪优秀人才支持计划(NECT-08-0731)资助项目

通讯作者: 姚斌, 高伟 E-mail: w_gao@bjfu.edu.cn; yaobin@mail.caas.net.cn

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引用本文:

李倩倩, 李中媛, 冯舵, 黄火清, 韩翠晓, 杨培龙, 姚斌, 高伟. 芽孢杆菌β-折叠桶植酸酶的原核可溶性表达优化及包涵体复性研究[J]. 中国生物工程杂志, 2012, 32(08): 49-55.

LI Qian-qian, LI Zhong-yuan, FENG Duo, HUANG Huo-qing, HAN Cui-xiao, YANG Pei-long, YAO Bin, GAO Wei. Optimizing Soluble Expression and Inclusion Body Renature Research of β-Propeller Phytase of Bacillus sp. HJB17 in E.coli. China Biotechnology, 2012, 32(08): 49-55.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I08/49

[1] Mullaney E J, Daly C B, Ullah A H. Advances in phytase research.Adv Appl Microbiol,2000(47):157-199.
[2] Pandey A,Szakacs G,Soccol C R,et al.Production,purification and properties of microbial phytases.Bioresource Technology,2001,77(3):203-214.
[3] Haefner S,Knietsch A,Scholten E,et al.Biotechnological production and applications of phytases.Appl Microbiol Biotechnol,2005,68(5):588-597.
[4] Bohn L,Meyer A S,Rasmussen S K.Phytate:impact on environment and human nutrition.A challenge for molecular breeding.J Zhejiang Univ Sci B,2008 9(3):165-191.
[5] Lei X,Porres J,Mullaney E,et al.Phytase:source,structure and application.In:Polaina J, MacCabo A P, Eds. Industrial Enzymes Structure, Function and Applications. Netherlands: Springer, 2007.505-529.
[6] Baruah K, Pal A K, Sahu N P,et al.Dietary protein level,microbial phytase,citric acid and their interactions on bone mineralization of Labeo-rohita (Hamilton) juveniles.Aquacult Res, 2005,36(8),803-812.
[7] Lei X G, Stahl C H.Biotechnological development of effective phytases for mineral nutrition and environmental protection.Appl Microbiol Biotechnol,2001,57(4):474-481.
[8] Ha N C,Oh B C,Shin S,et al.Crystal structures of a novel,thermostable phytase in partially and fully calcium-loaded states.Nature Struct Biol,2000,7:147-153.
[9] Fu S,Sun J,Qian L.Effect of Ca²⁺on beta-propeller phytases.Protein Pept Lett,2008,15(1):39-42.
[10] Huang H Q,Shao N,Wang Y R,et al.A novel beta-propeller phytase from Pedobacter nyackensi MJ11 CGMCC 2503 with potential as an aquatic feed additive.Appl Microbiol Biotechnol,2009,83(2):249-259.
[11] Bradford M M.A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem,1976,72: 248-254.
[12] Choi Y M,Suh H J,Kim J M.Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J Protein Chem,2001,20(4):287-292.
[13] Rao D E,Rao K V,Reddy V D.Cloning and expression of Bacillus phytase gene (phy)in Escherichia coli and recovery of active enzyme from the inclusion bodies.J Appl Microbiol,2008,105(4):1128-1137.
[14] Hartl F U,Hayer-Hartl M.Molecular chaperones in the cytosol from nascent chain to folded protein.Science,2002,295(5561):1852-1858.

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