共表达内质网响应蛋白HAC1对α-半乳糖苷酶在毕氏酵母中表达的影响

生物技术通报 ›› 2014, Vol. 0 ›› Issue (7): 184-189.

共表达内质网响应蛋白HAC1对α-半乳糖苷酶在毕氏酵母中表达的影响

钟开新, 陈丽芝, 李阳源

广东溢多利生物科技股份有限公司, 珠海 519060

收稿日期:2013-11-04 出版日期:2014-07-15 发布日期:2014-07-16
作者简介:钟开新, 男, 硕士, 研发助理, 研究方向：基因工程；E-mail：nhappy_z@163.com

Effect of Co-expressing Protein HAC1 on Expression of α-galactosidase in Pichia pastoris

Zhong Kaixin, Chen Lizhi, Li Yangyuan

Guandong VTR Bio-tech Co., LTD., Zhuhai 519060

Received:2013-11-04 Published:2014-07-15 Online:2014-07-16

摘要/Abstract

摘要： 为提高毕氏酵母（Pichia pastoris）中重组α-半乳糖苷酶的表达, 将来源于P. pastoris GS115的hac1基因构建至pPIC9K表达载体中, 转化到重组P. pastoris 中与α-半乳糖苷酶共表达。研究结果表明, 在AOX1启动子控制下, HAC1过表达提高了重组P. pastoris中α-半乳糖苷酶蛋白表达含量, 使α-半乳糖苷酶活性提高了2.2倍。经PCR产物鉴定, 结果表明重组P. pastoris基因组中插入hac1基因。经50 L发酵罐甲醇诱导168 h, Gal-HAC1-4#工程菌最终酶活达到6 560 U/mL, 比Gal-21#工程菌提高了27%。甲醇诱导后Gal-HAC1-4#发酵液中粗蛋白浓度均高于Gal-21#工程菌。表明共表达HAC1蛋白可提高毕氏酵母产α-半乳糖苷酶蛋白的能力。

关键词: HAC1, α-半乳糖苷酶, 毕氏酵母, 共表达, UPR反应

Abstract: To improve the production of a recombinant α-galactosidase in Pichia pastoris, the hac1 gene from P. pastoris GS115 constructed in pPic9K vector was coexpressed with α-galactosidase gene in P. pastoris X33. Results showed that the HAC1 protein under control of AOX1 promoter increased the expression level of α-galactosidase protein in P. pastoris. The α-galactosidase activity superexpressed with the HAC1 protein was up to 2.2 times than that of constitutive expression of HAC1. hac1 gene integrated into the genome of the P.pastoris was proved by PCR. The galactosidase activity of Gal-HAC1-4#, after 168 h methanol induction in 50 L high-density fermentation, reached the highest value of 6 560 U/mL, and increased by 27% compared with Gal-21#. The crude protein concentration of Gal-HAC1-4# was higher than the strain Gal-21# after methanol induced. These results showed that the galactosidase expression ability of Pichia pastoris was inproved by co-expressing protein HAC1.

Key words: HAC1, α-galactosidase, P. pastoris, Co-exprssion, UPR

钟开新, 陈丽芝, 李阳源. 共表达内质网响应蛋白HAC1对α-半乳糖苷酶在毕氏酵母中表达的影响[J]. 生物技术通报, 2014, 0(7): 184-189.

Zhong Kaixin, Chen Lizhi, Li Yangyuan. Effect of Co-expressing Protein HAC1 on Expression of α-galactosidase in Pichia pastoris[J]. Biotechnology Bulletin, 2014, 0(7): 184-189.

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