期刊文献+

重组人神经生长因子β亚基的原核优势表达及纯化 被引量:2

Overexpression and Purification of Recombinant Human Nerve Growth Factor β-Subunit in Escherichia coli
下载PDF
导出
摘要 目的:在原核系统内表达SUMO融合重组人神经生长因子β亚基(h NGF-β),并对其纯化。方法:将5'端引入了羟胺切割位点的h NGF-β基因克隆到表达载体p ET-SUMO中,IPTG诱导表达后,对表达的融合蛋白SUMO-h NGF-β包涵体进行亲和纯化,然后在变性条件下加入羟胺进行裂解,利用SUMO分子伴侣的功能与h NGF-β进行共复性,最后利用阳离子交换层析纯化获得重组h NGF-β。结果:融合蛋白SUMO-h NGF-β相对分子质量为39×103,表达量占细菌总蛋白的35%;在变性条件下经羟胺切割、共复性和阳离子纯化后,相对分子质量为14×103的重组h NGF-β复性率和纯度分别为30%和80%以上,Western印迹显示其具有良好的抗原特性。结论:h NGF-β与SUMO融合可以在原核表达系统中实现优势表达。 Objective:To acquire recombinant human nerve growth factor β-subunit(rhNGF-β) by fusing expression with small ubiquitin-related modifier(SUMO) in prokaryotic system and series of purification.Methods:The5' end modified hNGF-β gene was cloned to plasmid pET-SUMO to construct the fusion expression vector with a hydroxylamine cleavage site between hNGF- β and SUMO.The fusion protein was expressed in inclusion body and experienced affinity chromatography column,hydroxylamine cleavage under denatured condition,co- renaturation with SUMO protein,and SP-Sepharose Fast Flow.Results:The SUMO-hNGF-β fusion protein was expressed efficiently and stably in Escherichia coli,accounting for about 35%in total bacterial protein.The purity,M,and renaturation rate of final hNGF-β were 80%,14X103 and about 30%respectively,and its antigenicity was showed by Western blotting assay.Conclusion:Strategy of fusion expression with SUMO protein leads to high expression level rhNGF-β in prokaryotic system.
出处 《生物技术通讯》 CAS 2015年第6期776-779,共4页 Letters in Biotechnology
基金 国家传染病重大专项(2013ZX-10003-006)
关键词 入神经生长因子β亚基 SUMO蛋白 融合表达 共复性 human nerve growth factor β-subunit SUMO protein fusion expression co-renaturation
  • 相关文献

参考文献7

  • 1Levi-Montalcini R, Dal Toso R, Della Valle F, et al. Update of the NGF saga[J]. J Neurol Sci. 1995,130(2):119-127.
  • 2Swaisgood H E. The importance of disulfide bridging[J]. Biotechnol Adv. 2005,23:71-73.
  • 3Butt T R, Edavettal S C, Hall J P. et al.SUMO fuson tech- nology for difficult-to-express proteins[J]. Protein Expr Purif, 2005,43( 1 ): 1-4.
  • 4Dohmen H J. SUMO protein modification[J]. Biochim Biophys Aeta. 2004.1695(1-3):113-131.
  • 5Nelson J W, Creightom T E. Reactivity and ionizalion of the aetive site cysteine residues of DsbA. a protein reguired for disulfide bond formation in vivo[J]. Biochemistry, 1994,33: 5974-5983.
  • 6Joanne EM, Daniel CD, Kirk LH, et al. Calcium-acivated K + channels increase cell proliferation independent of K + conductance [J]. Am J Physiol Cell physiol, 2011,300(4) :792-802 .
  • 7Kast RE. Profound blockage of CXCR4 signaling at multiple points using the synergy between plerixafor, mirtazapine, and clotrimazole as a new glioblastoma treatment adjunct [ J ]. Turk Neurosurq, 2010,20(4) :425-429.

同被引文献21

引证文献2

二级引证文献5

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部