• 研究报告 •

### 人胃内因子在谷氨酸棒杆菌中的表达及活性研究

1. 粮食发酵工艺与技术国家工程实验室 工业生物技术教育部重点实验室江南大学 生物工程学院， 无锡 214122

• 出版日期:2020-04-18 发布日期:2020-04-17
• 通讯作者: 杨艳坤，副教授，硕士生导师，主要从事微生物学、分子生物学和发酵工程研究，E-mail：yangyankun@jiangnan.edu.cn；刘秀霞，副教授，硕士生导师，主要从事蛋白表达与合成生物学研究，E-mail：liuxiuxia@jiangnan.edu.cn；白仲虎，教授，博士生导师，主要从事发酵工程和生物医药过程工程等研究，E-mail：baizhonghu@jiangnan.edu.cn
• 作者简介:董贵彬，硕士，主要从事微生物表达系统研究，E-mail：donggb2012@163.com
• 基金资助:
国家自然科学基金（21808082，21878124）

### Expression and optimization of recombinant human gastric factors in Corynebacterium glutamicum

1. National Engineering Laboratory of Cereal Fermentation Technology, Key Laboratory of Industrial Biotechnology, School of Bioengineering, Jiangnan University, Wuxi 214122, China
• Online:2020-04-18 Published:2020-04-17

Abstract: The human gastric factor(GIF) is a very important protein, and it plays a very important role in assisting the absorption of VB12 through small intestinal wall cells. If there be synthetic disorder of the protein, it will lead to the occurrence of atypical anemia in the body and reduce immunity. C. glutamicum is a food-grade safe microorganism, no endotoxin is detected in the fermentation broth of C. glutamicum. In addition, it has some unique advantages in its use for protein expression. In order to express pharmaceutical GIF in C. glutamicum, a recombinant gene rgif was synthesized, and codon optimization of the gene sequences to ensure normal translation of rgif into GIF. The recombinant gene rgif was successfully cloned into the expression vector pXMJ19, the vector containing rgif was introduced into C. glutamicum, and the protein GIF was expressed successfully. To improve the yield of GIF, fermentation process was optimized through orthogonal experimental design. The optimal fermentation conditions to express GIF in C. glutamicum were as follows: IPTG concentration was 100 μmol/L, time of adding induction was 10 h, fermentation time was 22 h and fermentation temperature was 23 ℃. Under which, GIF was abundantly expressed in C. glutamicum, and it was soluble. Proteins harvested could be purified by HisTrap FF column. The protein could also be detected by an ELISA kit, and the yield of GIF reached to 42.3 mg/L. According to the results of orthogonal experiments, it was found that the yield of GIF continued to decrease as the fermentation temperature increased from 23 ℃ to 37 ℃. The yield of GIF was also continuously reduced during the time of adding induction from 0 to 10 h. When it comes to the fermentation time, GIF production increased continuously from 18 to 22 h, but decreased gradually after 22 h. As to IPTG, GIF production gradually increased as its concentration increased from 50 to 100 μmol/L, but decreased thereafter. The effect of the above four factors on protein production was weakened in turn. It could be seen that giving a lower temperature, delaying the time of induction would be an effective work to improve the soluble expression of GIF. This was the first report of the expression of GIF by prokaryote, which would provide a viable solution for prokaryotic expression of GIF.