Zhang Li, Jiang Yuanyuan, Zhang Jian, Yang Zhennai
Center of Agro-food Technology, Northeast Agricultural Research Center of China, Changchun 130033, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Aug;25(8):1160-5.
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.
为了在酵母中表达牛凝乳酶，我们通过PCR从质粒pMD18T-Prochy中扩增前凝乳酶基因，然后将该基因克隆到表达载体pPICZaA中，得到pPICZaA-Prochy。毕赤酵母GS115用作宿主细胞。通过PCR和测序分析确认前凝乳酶cDNA整合到毕赤酵母基因组中。凝乳酶在毕赤酵母中成功表达，SDS-PAGE显示在约37 kD处有一条强条带。活性测试表明，培养上清液的凝乳酶活性为12.2 SU/mL。这是凝乳酶在毕赤酵母中成功表达的首次报道。本研究获得的重组毕赤酵母菌株可进一步用于生产用于奶酪制作的重组凝乳酶。
