Recombinant Expression And Activity Identification Of Brevinine-2GUB Peptide From The Extract Of Asian Frog's Skin

With the development of economic and people's living standards,diabetes mellitus has become one of the most common chronic diseases worldwide.According to the International Diabetes Federation(IDF),there were 451 million(18-99 years old)diabetic patients worldwide in 2017,and the number of people with diabetes is expected to increase to 693 million by 2045,which 80%-90% belong to type II diabetes mellitus.Since the current clinical treatments for type II diabetes mellitus,such as sulfonylureas,metformin and glinide,have limited efficacy,tolerance and toxic side effects,it is necessary to develop bioactive peptides which regulate glucose metabolism and significantly reduce side effects.The Brevinin-2GUb peptide is a peptide with antibacterial activity and significant insulin-releasing activity which was isolated from the skin of Asian frog Hylarana guntheri,which showed great research value.The recombinant plasmid pET32a-Trx-His-EK-Brevinin-2GUb was used to express the Trx-His-EK-Brevinin-2GUb fusion protein in Escherichia coli.Through optimization of its expression temperature,it showed that the fusion protein had the highest yield with 36 mg/L while inducing at 16 ? for 20 hours.The Trx-His-EK-Brevinin-2GUb fusion protein was successfully cleaved by enterokinase(EK).Then purified Brevinin-2GUb with any tag was obtained with a yield of approximate 2.5 mg/L and the purity ?90%.The purified Brevinin-2GUb was identified by RP-HPLC,circular dichroism spectroscopy and liquid chromatography tandem mass spectrometry.The results showed that the amino acid sequence of purified Brevinin-2GUb was consistent with the synthesized Brevinin-2GUb.However,the?-helix structure of the purified Brevinin-2GUb was lower than that of the synthetic Brevinin-2GUb with disulfide.The mass spectrometry results indicated that the purified Brevinin-2GUb formed a disulfide bond but the formation rate of disulfide bonds did not reach 100%.The purified Brevinin-2GUb peptide had no hemolytic activity toward mammalian cells at the concentration of 170 ?g/mL,and it exhibited significant antibacterial activity against Bacillus megaterium ATCC 14945(MIC=10.63 ?g/mL).In addition,purifiedBrevinin-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 ?g/mL.In this study,the antibacterial activity and insulin-releasing activity of systhetic Brevinin-2GUb without disulfide were compared to systhetic Brevinin-2GUb with disulfide.The results demonstrated that the antibacterial activity and insulin-releasing activity of Brevinin-2GUb without disulfide were weaker than that of Brevinin-2GUb with disulfide,which indicated that the disulfide bond was very important to the biological activity of Brevinin-2GUb.Finally,the cytotoxicity of Brevinin-2GUb peptide was determinated by the MTT assay against Vero,HepG2,HT-29 and Hela cell lines.The results showed that the Brevinin-2GUb peptide had significant cytotoxic effects against HT-29 cell lines.Therefore,Brevinin-2GUb peptide has a good application prospect in the treatment of diabetes mellitus and cancer.