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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (11): 58-63    
研究报告     
乙酰氨基半乳糖转移酶14(GALNT14)在毕赤酵母中的表达纯化及活性鉴定
吴琛1, 葛建峰1, 张博1, 马思思2, 刘芳铭1, 王媛媛1, 田焕娜1, 刘晓波2, 张晓康1, 李秦剑1
1. 河北大学生命科学学院 保定 071002;
2. 河北大学药学院 保定 071002
Expression,Purification and Activity Analysis of Human GALNT14 in Pichia Pastoris
WU Chen1, GE Jian-feng1, ZHANG Bo1, MA Si-si2, LIU Fang-ming1, WANG Yuan-yuan1, TIAN Huan-na1, LIU Xiao-bo2, ZHANG Xiao-kang1, LI Qin-jian1
1. College of Life Sciences,Hebei University,Baoding 071002,China;
2. College of Pharmacy,Hebei University,Baoding 071002,China
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摘要:

将GALNT14全长编码区克隆到分泌型酵母表达载体pPIC9K中,构建重组载体pPIC9K-T14,经电转至毕赤酵母GS115中表达,使用G418筛选高表达重组菌,并对诱导条件进行优化,表达产物使用SDS-PAGE分析、Western blot鉴定、Sephadex G-100纯化、最后经HPLC检测活性。结果显示,在提供更好的供氧量条件下,用0.75%甲醇诱导可提高目的蛋白的表达量而降低杂蛋白的表达。培养上清液经SDS-PAGE检测显示目的蛋白分子量约64 kDa;Western blot结果显示在相应分子量处有一条特异性条带;利用Sephadex G-100成功分离纯化了目的蛋白;活性测定结果显示所获得的目的蛋白具有催化活性,为深入研究GALNT14的结构与功能奠定了基础。

关键词: GALNT14毕赤酵母分泌表达纯化活性测定    
Abstract:

The full-length cDNA of GALNT14 was cloned into the pPIC9K vector. The recombinant vector was transformed into Pichia pastoris GS115 using electroporation. The recombinant strains with high expression of GALNT14 were screened using G418. Different methanol concentration was applied to optimize the cultivation conditions. The expressed recombinant protein was detected by SDS-PAGE and Western blotting,then purified by Sephadex G-100. The activity of recombinant protein was analyzed with HPLC. The result showed that the protein production was increased with better oxygen supply in the cultivation and 0.75% methanol. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 64 kDa. Western blotting analysis suggested that it was human GALNT14 protein. Enzyme activity assay showed the purified GALNT14 has glycosyltransferase activities, which provide a basis for the further study on the function and structure of the GALNT14.

Key words: GALNT14    Pichia pastoris    Secreting expression    Purification    Activity analysis
收稿日期: 2011-06-16 出版日期: 2011-11-25
ZTFLH:  Q78  
基金资助:

国家自然科学基金(30800180)、河北省自然科学基金(C2009000191)、河北省教育厅科学研究计划 (2008-106)资助项目

通讯作者: 吴琛     E-mail: dawnwuchen@163.com
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引用本文:

吴琛, 葛建峰, 张博, 马思思, 刘芳铭, 王媛媛, 田焕娜, 刘晓波, 张晓康, 李秦剑. 乙酰氨基半乳糖转移酶14(GALNT14)在毕赤酵母中的表达纯化及活性鉴定[J]. 中国生物工程杂志, 2011, 31(11): 58-63.

WU Chen, GE Jian-feng, ZHANG Bo, MA Si-si, LIU Fang-ming, WANG Yuan-yuan, TIAN Huan-na, LIU Xiao-bo, ZHANG Xiao-kang, LI Qin-jian. Expression,Purification and Activity Analysis of Human GALNT14 in Pichia Pastoris. China Biotechnology, 2011, 31(11): 58-63.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I11/58


[1] Wang H, Tachibana K, Zhang Y, et al.UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer maker.J Histochem Cytochem,2006,54:317-328.

[2] Rao J S. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer,2003, 3(7): 489-501.

[3] 仇灏,吴士良,陈克平,等. 多肽:N-乙酰氨基半乳糖转移酶2(Polypeptide:N-Acetylgalactosaminyltransferases,pp-GalNAc-T2,E.C.2.4.1.41)的mRNA表达谱研究.中国血液流变学杂志,2003,13(1):24-25. Qiu H,Wu S L,Chen K P,et al. Chinese Journal of Hemorheology,2003,13(1):24-25.

[4] Shibao K, Izumi H, Nakayama Y, et al. Expression of UDP-N-acetyl-alpha-D-galactosamine-polypeptide galNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma. Cancer, 2002,94(7): 1939-1946.

[5] Berois N, Mazal D, Ubillos L, et al. UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker. J.Histochem.Cytochem, 2006, 54(3): 317-328.

[6] 莫建华,吴士良,陈克平,等.不同白血病细胞N-乙酰氨基半乳糖转移酶Ⅰ-ⅦmRNA的表达.中国血液流变学杂志,2004,14(2):173-177. Mo J H,Wu S L,Chen K P,et al. Chinese Journal of Hemorheology,2004,14(2):173-177.

[7] Wu C, Guo X D, Wang W N, et al.N-Acetylgalactosaminyltransferase-T14 as a potential biomarker for breast cancer by immunohistochemistry.BMC Cancer,2010,10(1):123.

[8] Cregg J M, Cereghino J L, Shi J, et al. Recombinant proteinexpression in Pichia pastoris. Mol Biotechnol, 2000, 16(1): 23-52.

[9] Brockhausen I. Pathway of O-glycan biosynthesis in cancer cells. Biochem Biophys Acta, 1999,1473(1):67-95.

[10] Hagen F K, Hazes B, Raffo B, et al.Structure-function analysis of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.J Biol Chem,1999,274(10):6797-6803.

[11] Tenmo M, Saeki A, Kezdy F J. The lectin domain of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites. J Biol Chem, 2002, 277(49): 47088-47096.

[12] Milac A L, Buchete N V, Fritz T A, et al.Substrate-induced conformational changes and dynamics of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase-2.J Mol Biol,2007,373(2):439-451.

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