牛凝乳酶基因在毕赤酵母中的重组表达

Recombinant expression of bovine chymosin in Pichia pastoris

摘要:

通过PCR技术从克隆载体pMD18T-Prochy上扩增牛凝乳酶原基因, 双酶切后定向插入到酵母表达载体pPICZaA中,构建表达质粒pPICZaA-Prochy, 线性化后电转化毕赤酵母GS115, 经PCR和测序鉴定凝乳酶原基因成功插入到毕赤酵母的基因组中。在甲醇诱导下进行凝乳酶的表达, SDS-PAGE分析证明重组凝乳酶的分子量约为37 kD, 培养基上清液中凝乳酶的活性为12.2 SU/mL。本研究首次应用毕赤酵母表达牛凝乳酶, 在培养基中获得分泌表达的重组凝乳酶, 为干酪工业提供了新型及优良的凝乳酶来源。

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.