Over-expression purification and isotopic labeling of a tag-less human glucose-dependent insulinotropic polypeptide (hGIP)

摘要

Glucose-dependent insulinotropic polypeptide (GIP), a gut peptide released in response to food intake brings about secretion of insulin in a glucose-dependent manner upon binding to its receptor, GIPR. GIP–GIPR has emerged as a new vista for anti-diabetic drug discovery and their interaction is being probed at the atomic level to aid rational drug design. In order to probe this interaction on cells, the current study attempts towards expressing ¹⁵N-labeled GIP using classical molecular biology tools. We have developed a methodology to obtain GIP devoid of extra amino acid(s); a prerequisite to the intended interaction study. The synthetic GIP cDNA with a Factor Xa protease site at the N-terminus of GIP was inserted in the vector pET32a(+); the fusion protein thus expressed was eventually cleaved to obtain GIP. After successful Factor Xa cleavage, the cleaved GIP was confirmed by western blot. Subsequently, the (¹⁵N)GIP was obtained using the aforementioned procedure and confirmed by MALDI-TOF.