摘要
为实现降糖多肽Brevinin-2GUb在大肠杆菌中的高效表达,本文将Brevinin-2GUb基因插入到含有硫氧还蛋白、His标签及肠激酶酶切位点的p ET32a载体中,将重组表达载体p ET32a-Trx-His-EK-Brevinin-2GUb转化至大肠杆菌中诱导表达Trx-His-EK-Brevinin-2GUb融合蛋白。通过对表达温度进行优化,结果显示16℃诱导表达20 h后融合蛋白产量达到36 mg/L;该融合蛋白经肠激酶(EK)特异性切割后成功获得不带标签的Brevinin-2GUb多肽,产量约为2.50 mg/L,纯度达90%以上。通过反相高效液相色谱对重组表达的Brevinin-2GUb进行鉴定,结果显示重组表达的Brevinin-2GUb与人工合成的Brevinin-2GUb的氨基酸序列一致。通过血平板检测发现,170μg/m L的重组表达Brevinin-2GUb多肽无溶血效应。此外,重组表达的Brevinin-2GUb多肽在浓度为10μg/m L时对INS-1细胞产生显著的刺激胰岛素分泌作用(为基础释放量的120.04%;p<0.01)。研究结果为Brevinin-2GUb多肽开发为降糖口服液及多肽药物奠定了基础。
In order to achieve high expression of the hypoglycemic peptide Brevinin-2 GUb in E. coli, the Brevinin-2 GUb gene was inserted into the p ET32 a vector containing thioredoxin, His tag and enterokinase cleavage site, and then the recombinant expression vector p ET32 a-Trx-His-EK-Brevinin-2 GUb was obtained, which was transformed into E. coli for the fusion protein Trx-His-EK-Brevinin-2 GUb expression. Through optimization of its expression temperature, results showed that the fusion protein had the highest yield with 36 mg/L after inducing at 16 ℃ for 20 hours. The Trx-His-EK-Brevinin-2 GUb fusion protein was successfully cleaved by enterokinase(EK). The purified Brevinin-2 GUb without any tag was obtained with a yield of approximate 2.50 mg/L and the purity ≥90%. The purified Brevinin-2 GUb was identified by reversed phase high performance liquid chromatography. The results showed that the amino acid sequence of purified Brevinin-2 GUb was consistent with the synthesized Brevinin-2 GUb. The purified Brevinin-2 GUb peptide had no hemolytic activity toward mammalian cells at the concentration of 170 μg/m L. In addition, purified Brevinin-2 GUb produced a significant stimulation of insulin release(120.04% of basal rate;p<0.01) against INS-1 cell lines at a concentration of 10 μg/m L. The results can provide the foundation for the development of Brevinin-2 GUb peptide as a hypoglycemic oral solution and a peptide drug.
作者
林静莲
黄敏华
王蒙
马毅
王菊芳
LIN Jing-lian;HUANG Min-hua;WANG Meng;MA Yi;WANG Ju-fang(School of Bioscience and Bioengineering,South China University of Technology,Guangzhou 510006,China)
出处
《现代食品科技》
EI
CAS
北大核心
2020年第5期155-162,共8页
Modern Food Science and Technology
基金
科技部“863”科技计划项目(2014AA021004)。
