重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

中国生物工程杂志

2012, Vol. 32

Issue (05): 24-30

研究报告

重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

孔蕴¹, 郜海涛¹, 李树芳¹, 王鹏^1,2, 顾国锋², 顾黎^1,2

1. 山东大学生命科学学院微生物技术国家重点实验室济南 250100;
2. 国家糖工程技术研究中心济南 250100

Expression and Purification of Functional HuGALNT3 without the Transmembrane Domain (huGALNT3-sol) in Pichia pastoris

KONG Yun¹, GAO Hai-tao¹, LI Shu-fang¹, WANG Peng^1,2, GU Guo-feng², GU Li^1,2

1. State key laboratory for Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China;
2. National Glycoengineering Research Center, Jinan 250100, China

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摘要：

目的:为了获得有催化活性的人乙酰半乳糖胺转移酶3 (GALNT3),构建了GALNT3可溶性区域(GALNT3-sol)的真核分泌表达载体,在巴斯德毕赤酵母中表达并纯化GALNT3-sol蛋白,体外检测其转糖基活性。方法:以构建好的pET15b/GALNT3-sol为模板进行PCR,扩增编码人GALNT3-sol的cDNA片段(1 755 bp),将其克隆至真核表达载体pPIC9K,载体线性化后采用电击法转化毕赤酵母 GS115。通过MD平板和G418平板筛选出阳性高拷贝重组菌株。阳性菌株经过甲醇诱导表达人GALNT3-sol重组蛋白,表达上清进行Ni-NAT分离纯化。分别采用SDS-PAGE和Western blot鉴定纯化的重组蛋白,并使用HPLC和MALDI-TOF/MS分析其转糖基化反应的活性。结果:成功构建了能够分泌表达GALNT3-sol的毕赤酵母菌株。阳性表达菌株在 BMMY 培养基 (pH 6.0) 中 20℃培养,经 0.5％甲醇诱导表达 96 h,摇瓶表达量可达 5mg/L。SDS-PAGE和Western blot结果显示表达重组蛋白为糖基化形式。活性检测显示表达的重组蛋白具有转糖基活性。结论:成功获得可以高效分泌表达具有活性的人GALNT3-sol蛋白的毕赤酵母菌株,为进一步研究人GALNT3的性质及其应用提供了基础。

关键词： GALNT3; 毕赤酵母; 分泌表达; 活性

Abstract:

Objective:In order to research the bioactivity of GALNT3, the truncated part of GALNT3 (huGALNT3-sol) which was deleted of the hydrophobic trans-membrane domain were obtained using Pichia pastoris expression system, and assayed the transferring GalNAc activity of recombinant huGALNT3-sol. Methods: The gene of human GALNT3-sol (1 755 bp)was amplified from pET15b/ GALNT3-sol and cloned into expression vector pPIC9k, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains with high-level huGALNT3-sol production were screened out by MD plate and G418. High level of huGALNT3-sol was obtained in BMMY medium the induction of methanol, and purified from the supernatant with Ni-NAT.The identity of the recombinant protein was confirmded by SDS-PAGE and then Western blotting analysis. HPLC and MALDI-TOF-MS analysis were used to identify the bioactivity of recombinant huGALNT3-sol. Results:The recombinant Pichia pastoris which could secretory express the human GALNT3-sol protein was constructed successfully. High level of huGALNT3-sol was obtained in BMMY medium (pH 6.0) after 96 hours induction of 20℃ and 0.5% methanol, with the highest yield of 5mg/L in shake flask culture. The identity of the recombinant protein was confirmed by Western blot analysis and the huGALNT3-sol expressed in Pichia pastoris is in higher molecular weight glycoforms. The activity assay showed the recombinant huGALNT3-sol has the activity of transferring GalNAc to Ser/Thr residues in peptide. Conclusion:The human GALNT3-sol, which has the activity of transferring GalNAc to Ser/Thr residues in peptide, was successfully expressed and purified in Pichia pastoris. It provides support for the further research and application of GALNT3.

Key words: GALNT3 Pichia pastoris Secretory expression Activity

收稿日期: 2012-01-13 出版日期: 2012-05-25

ZTFLH:

Q786

基金资助:

国家自然科学基金青年资助项目(31000368)

通讯作者: 顾黎 E-mail: yehe.gl@sdu.edu.cn

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引用本文:

孔蕴, 郜海涛, 李树芳, 王鹏, 顾国锋, 顾黎. 重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定[J]. 中国生物工程杂志, 2012, 32(05): 24-30.

KONG Yun, GAO Hai-tao, LI Shu-fang, WANG Peng, GU Guo-feng, GU Li. Expression and Purification of Functional HuGALNT3 without the Transmembrane Domain (huGALNT3-sol) in Pichia pastoris. China Biotechnology, 2012, 32(05): 24-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I05/24

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