RESUMO
Tandem repeat multimers of Momordica charantia (MC) peptide MC6 were designed and the recombinant plasmid containing 10 copies of MC6 gene was constructed to improve the expression level of MC6 in Escherichia coli. Under the selected conditions of cultivation and induction, the expression level of recombinant TrxA-MC6(10) protein was above 25% of total bacteria protein. This fusion protein was purified and cleaved with HCl (13%, w/v). Either the un-cleaved or cleaved recombinant proteins was analyzed pharmacological activity by alloxan-induced diabetic mice and only the cleaved products of the recombinant protein showed significant hypoglycemic effects. The study provides a convenient and economical method for the large-scale production of anti-diabetic medicines for pharmaceutical applications.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/metabolismo , Momordica charantia/química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Aloxano , Animais , Glicemia/análise , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dosagem de Genes , Expressão Gênica , Ácido Clorídrico/química , Hidrólise , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Camundongos , Peptídeos/genética , Peptídeos/farmacologia , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Transformação BacterianaRESUMO
BACKGROUND: Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. RESULTS: In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. CONCLUSION: This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.