vMIP-II-TfN融合蛋白在毕赤酵母中的表达和纯化

中国生物工程杂志

2011, Vol. 31

Issue (10): 23-28

研究报告

vMIP-II-TfN融合蛋白在毕赤酵母中的表达和纯化

王峰, 孙晗笑, 李秀英, 张光, 莫雪梅

暨南大学药学院基因组药物研究所广州 510632

Expression and Purification of vMIP-II-TfN in Pichia pastoris

WANG Feng, SUN Han-xiao, LI Xiu-ying, ZHANG Guang, MO Xue-mei

Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China

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摘要：

目的:构建pPIC-vMIP-II-TfN酵母表达载体,表达纯化vMIP-II-TfN融合蛋白。方法:利用PCR方法扩增编码人转铁蛋白N端半分子的基因片段,通过酶切、连接、转化等分子克隆方法构建pPIC-vMIP-II-TfN酵母表达载体;电击法转化X33酵母菌;用甲醇诱导重组酵母菌表达融合蛋白,利用硫酸铵沉淀、透析、Ni-NTA层析等技术进行蛋白纯化,SDS-PAGE和Western blot检测蛋白表达和纯化情况,利用趋化实验进行纯化蛋白活性检测。结果:经过两次PCR扩增了一个长约1.1kb的包含Xba I酶切位点的IgG3-TfN基因片段,插入pPIC-vMIP-II的Xba I酶切位点,经菌液PCR鉴定获得重组子,测序结果表明构建载体pPIC-vMIP-II-TfN的表达框正确无误,转化X33酵母菌,用甲醇诱导表达出48kDa的vMIP-II-TfN融合蛋白,经硫酸铵沉淀、透析、Ni-NTA纯化后得到纯度约为95%的vMIP-II-TfN融合蛋白。Western印迹结果表明融合蛋白能与转铁蛋白抗体特异性结合。活性检测表明经过诱导表达的vMIP-II-TfN融合蛋白具有趋化抑制活性。结论:成功构建pPIC-vMIP-II-TfN酵母表达载体,重组酵母工程菌经甲醇诱导成功表达出vMIP-II-TfN融合蛋白,纯化后的vMIP-II-TfN融合蛋白具有趋化抑制活性。

关键词： vMIP-II; 转铁蛋白; 蛋白表达; 纯化

Abstract:

Objective: To construct the pPIC-vMIP-II-TfN yeast expression vector and to express vMIP-II-TfN fusion protein. Method: PCR was employed to amplify the N-terminal half-molecular of human transferrin gene. Molecular cloning methods including digestion, ligation and transformation were used to construct the pPIC-vMIP-II-TfN yeast expression vector. The linearized pPIC-vMIP-II-TfN was transformed into Pichia pastoris X33 strain by electroporation. The transformant was induced by methanol to express target protein and ammonium sulfate precipitation, dialysis, and Ni-NTA affinity chromatography were used to purify the target protein. SDS-PAGE and Western blot was used to detect the expression and purification of vMIP-II-TfN. The activity of vMIP-II-TfN was detected by chemotaxis inhibition activity assay. Results: A 1.1kb IgG3-TfN DNA fragment containing the Xba I site was amplified and it was inserted into Xba I sites of pPIC-vMIP-II. After PCR identification and sequencing, the recombinant plasmid pPIC-vMIP-II-TfN was obtained successfully and the open reading frame was correctly. The linearized pPIC-vMIP-II-TfN by Sac I was transformed into Pichia pastoris X33 strain. After inducing with methanol for 72 h, the transformant was confirmed to express a recombinant protein with molecular mass of 48 kDa by SDS-PAGE and Western blot. After characterization, the vMIP-II-TfN fusion protein was successfully purified from the supernatant of the broth using ammonium sulfate precipitation, dialysis and affinity chromatography. Furthermore, in vitro bioacitivity assay indicated that vMIP-II-TfN has chemotaxis inhibition activity. Conclusion: The pPIC-vMIP-II-TfN yeast expression vector was constructed successfully. The transformant can express the vMIP-II-TfN fusion protein which had chemotaxis inhibition activity.

Key words: vMIP-II Transferrin Protein expression Purification

收稿日期: 2011-06-29 出版日期: 2011-10-25

ZTFLH:

Q789

基金资助:

国家自然科学基金资助项目(30700704)

通讯作者: 孙晗笑 E-mail: novelfunction@163.com

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引用本文:

王峰, 孙晗笑, 李秀英, 张光, 莫雪梅. vMIP-II-TfN融合蛋白在毕赤酵母中的表达和纯化[J]. 中国生物工程杂志, 2011, 31(10): 23-28.

WANG Feng, SUN Han-xiao, LI Xiu-ying, ZHANG Guang, MO Xue-mei. Expression and Purification of vMIP-II-TfN in Pichia pastoris. China Biotechnology, 2011, 31(10): 23-28.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I10/23

[1] Shao W, Fernandez E, Sachpatzidis A, et al. CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure. Eur J Biochem, 2001, 268(10):2948-2959.

[2] Kledal T N, Rosenkilde M M, Coulin F, et al. A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus. Science, 1997, 277:1656-1659.

[3] Holst P J, Luttichau H R, Schwartz T W, et al. Virally encoded chemokines and chemokine receptors in the role of viral infections. Contrib Microbiol, 2003, 10:232-252.

[4] Morris K V, Higgins J, Shen X, et al. The effects of HHV-8 vMIP-II on SIVmac251 infection and replication competent and incompetent SIVmac239Delta3 vectors. Virus Research, 2003, 94:103-112.

[5] 莫雪梅, 叶石敦, 张光, 等. 广谱趋化因子受体结合物-vMIP-II的体内抗SIV功能研究. 中国病毒学, 2005, 20:459-463. Mo X M, Ye S D, Zhang G, et al. Virologica Sinica, 2005, 20:459-463.

[6] Widera A, Norouziyan F, Shen W C. Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery. Adv Drug Deliv Rev, 2003, 55(11):1439-1466.

[7] Bai Y, Ann D K, Shen W C. Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent. Proc Natl Acad Sci USA, 2005, 102(20):7292-7296.

[8] Sallach R E, Conticello V P, Chaikof E L. Expression of a recombinant elastin-like protein in Pichia pastoris. Biotechnol Prog, 2009, 25(6):1810-1818.

[9] Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology, 1993, 11(8):905-910.

[10] Daly R, Hearn M T. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit, 2005, 18(2):119-138.

[11] Pack P, Pluckthun A. Mini-antibodies: use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli. Biochemistry, 1992, 31(6):1579-1584.

[12] Lambert L A, Perri H, Meehan T J. Evolution of duplications in the transferrin family of proteins. Comp Biochem Physiol B Biochem Mol Biol, 2005, 140(1):11-25.

[13] Steinlein L M, Graf T N, Ikeda R A. Production and purification of N-terminal half-transferrin in Pichia pastoris. Protein Expr Purif, 1995, 6(5):619-624.

[14] Xia C Q, Wang J, Shen W C. Hypoglycemic effect of insulin-transferrin conjugate in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther, 2000, 295(2):594-600.

[15] 李晓静, 张豪, 薛冲, 等. 干扰素与转铁蛋白融合蛋白在毕赤酵母中的表达及鉴定. 生物化学与生物物理进展, 2005, 32(7):625-629. Li X J, Zhang H, Xue C, et al. Progress in Biochemistry and Biophysics, 2005, 32(7):625-629.

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