生物工程學報 25 August 2009, 25(8):1160～1165

牛凝乳酶基因在畢赤酵母中的重組表達

張莉, 姜媛媛, 張健, 楊貞耐

中國農(nóng)業(yè)科技東北創(chuàng)新中心農(nóng)產(chǎn)品加工研究中心, 長春 130033

摘  要: 通過PCR技術(shù)從克隆載體pMD18T-Prochy上擴增牛凝乳酶原基因, 雙酶切后定向插入到酵母表達載體pPICZaA中,構(gòu)建表達質(zhì)粒pPICZaA-Prochy, 線性化后電轉(zhuǎn)化畢赤酵母GS115, 經(jīng)PCR和測序鑒定凝乳酶原基因成功插入到畢赤酵母的基因組中。在甲醇誘導(dǎo)下進行凝乳酶的表達, SDS-PAGE分析證明重組凝乳酶的分子量約為37 kD, 培養(yǎng)基上清液中凝乳酶的活性為12.2 SU/mL。本研究首次應(yīng)用畢赤酵母表達牛凝乳酶, 在培養(yǎng)基中獲得分泌表達的重組凝乳酶, 為干酪工業(yè)提供了新型及優(yōu)良的凝乳酶來源,。

關(guān)鍵詞: 牛凝乳酶, 分泌表達, 畢赤酵母

Recombinant expression of bovine chymosin in Pichia pastoris

Li Zhang, Yuanyuan Jiang, Jian Zhang, and Zhennai Yang

Center of Agro-food Technology, Northeast Agricultural Research Center of China, Changchun 130033, China

Abstract: To express bovine chymosin in yeast, we amplified the prochymosin gene from the plasmid pMD18T-Prochy by PCR, and then cloned the gene into the expression vector pPICZaA, resulting in pPICZaA-Prochy. Pichia pastoris GS115 was used as host cells. Integration of the prochymosin cDNA into the Pichia pastoris genome was confirmed by PCR and sequencing analysis. Chymosin was expressed in Pichia pastoris successfully, and a strong band at about 37 kD was shown by SDS-PAGE. Activity tests showed that the chymosin activity of the culture supernatant was 12.2 SU/mL. This is the first report of successful expression of chymosin in Pichia pastoris. The recombinant Pichia pastoris strain obtained in this study could be further used to produce recombinant chymosin for cheese making.

Keywords: bovine chymosin, secretory expression, Pichia pastoris

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