Gene Cloning And Characterization Of Hypoglycemic Polypeptide-P From Momordica Charantia

Polypeptide-P is a 166 amino acid residues polypeptide, which exists in the seeds of Momordica charantia (MC). Previous studies have been reported that polypeptide-P has hypoglycemic effect. Although this peptide has been purified and amino acid sequence was determined before, the gene sequence has not been demonstrated yet.In this study, using the strategies of the degenerate RT-PCR and genome walking, a total of 498 bp cDNA involving the mature polypeptide-P has been cloned from MC. The deduced amino acid sequence of this cloned cDNA was identified as the polypeptide-P and has no homology to any of the known proteins in the database. The recombinant of polypeptide-P was successfully expressed with pET32a vector in Escherichia coli, purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE. The molecular weight of recombinant protein is approximately 44 kDa and 90% of the fusion protein was found to be produced in the soluble form. Furthermore the recombinant polypeptide was used in treatment on alloxan-induced diabetic mice. The results showed that the recombinant polypeptide has a significant hypoglycemic effect (P<0.01) and the fused tag has no effect on its biological function.The study of cloning, expression, purification and characterization of hyperglycaemic polypeptide-P proved that the recombinant polypeptide also has hypoglycemic effect and this would give a way to develop new drugs and produce in large scale.