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| 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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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.
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Received: 27 April 2012
Published: 25 August 2012
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[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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