Abstract:In order to achieve high expression of the hypoglycemic peptide Brevinin-2GUb in E. coli, the Brevinin-2GUb gene was inserted into the pET32a vector containing thioredoxin, His tag and enterokinase cleavage site, and then the recombinant expression vector pET32a-Trx- His-EK-Brevinin-2GUb was obtained, which was transformed into E. coli for the fusion protein Trx-His-EK-Brevinin-2GUb 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-2GUb fusion protein was successfully cleaved by enterokinase (EK). The purified Brevinin-2GUb without any tag was obtained with a yield of approximate 2.50 mg/L and the purity ≥90%. The purified Brevinin-2GUb was identified by reversed phase high performance liquid chromatography. The results showed that the amino acid sequence of purified Brevinin-2GUb was consistent with the synthesized Brevinin-2GUb. The purified Brevinin-2GUb peptide had no hemolytic activity toward mammalian cells at the concentration of 170 μg/mL. In addition, purified Brevinin-2GUb 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/mL. The results can provide the foundation for the development of Brevinin-2GUb peptide as a hypoglycemic oral solution and a peptide drug.
