毕赤酵母<em>STE13</em>基因敲除对GGH表达及其生物活性的影响

中国生物工程杂志

2013, Vol. 33

Issue (4): 28-33

研究报告

毕赤酵母STE13基因敲除对GGH表达及其生物活性的影响

路庆鹏, 许正宏, 史劲松, 窦文芳

江南大学药学院制药工程实验室无锡 214122

The Disruption of STE13 Gene in Pichia pastoris Improves the Expression and Bioactivity of GGH

LU Qing-peng, XU Zhen-hong, SI Jin-song, DOU Wen-fang

Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China

全文: PDF(670 KB) HTML

摘要： 目的:重组毕赤酵母GS115/pPIC9K-GGH表达的人胰高血糖素样肽-1-人血清白蛋白融合蛋白[(GLP-1 A₂G )₂-HSA,GGH]的细胞活性测评结果显示生物活性较低,这可能与其表达过程中蛋白降解有一定关联。通过对毕赤酵母GS115 STE13 基因的敲除,获得一株完整表达蛋白GGH的毕赤酵母宿主,并进一步提高其表达产物GGH的生物活性。方法:采用基于PCR技术介导的Cre-Loxp系统重组技术成功敲除宿主菌GS115的STE13 基因,得到一株STE13 缺陷型菌株GS115/D13,并通过细胞活性测评系统检测GS115/D13表达产物GGH的生物活性。结果:通过对STE13 基因缺陷型菌株GS115/D13与出发菌株GS115/W发酵表达对比,显示GS115/D13菌株表达融合蛋白GGH生物活性有明显提升,且表达量提高了25.8%,两菌株生长无明显差异。结论:STE13 基因的敲除成功缓解了GGH表达过程中的降解问题,并相对出发菌株GS115/W大幅度提高了蛋白生物活性。

关键词： 毕赤酵母; GLP-1; STE13; 生物活性

Abstract: Objective: It is barely found that the bioactivity of recombination protein (GLP-1 A₂G )₂-HSA (GGH) by the cell bioactivity evaluation that related to its protein degradation, which expressed in methylotrophic yeast Pichia pastoris GS115 strain. Therefore, A new P.pastoris GS115 strain with STE13 gene disruption was engineered, helped for the expression of intact GGH, and the bioactivity can be further improved. Methods: The STE13 gene deficient strain GS115/D13 was constructed by the disruption of the P.pastoris homolog of the Saccharomyces cereviaiae dipeptidyl aminopeptidase (STE13 ) gene in Pichia genome by the Cre-Loxp recombination system based on the fusion PCR technology, and the bioactivity of GGH estimated by the cell biological evaluation methods. Results: The recombinant strain GS115/D13 showed the protein degradation much more relieved, apparent bioactivity improved and the yield of about 25.8% increased compared with that in the wild-type strain (GS115/W). Conclusion: The results suggested that STE13 gene disruption offered an expression host for the product of much more intact GGH and helped improve the protein bioactivity.

Key words: Pichia pastoris GLP-1 STE13 Bioactivity

收稿日期: 2013-01-14 出版日期: 2013-04-25

ZTFLH:

Q819

基金资助: 国家自然科学基金(31000016);江苏省科技支撑计划(BE2009629)

通讯作者: 窦文芳 E-mail: douwenfang@yahoo.com.cn

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引用本文:

路庆鹏, 许正宏, 史劲松, 窦文芳. 毕赤酵母STE13基因敲除对GGH表达及其生物活性的影响[J]. 中国生物工程杂志, 2013, 33(4): 28-33.

LU Qing-peng, XU Zhen-hong, SI Jin-song, DOU Wen-fang. The Disruption of STE13 Gene in Pichia pastoris Improves the Expression and Bioactivity of GGH. China Biotechnology, 2013, 33(4): 28-33.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/28

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