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

Rakesh C Chandarana,Evans C Coutinho,Jacinta S D’Souza,Anil Saran,Ashok K Varma,Vikrant Vikrant

doi:10.1007/s13205-013-0181-x

Abstract

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.

Highlights

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
Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone released into the blood stream in response to glucose and nutrients absorption from the intestine along with glucagon like polypeptide-1 (GLP-1) (Drucker 2006; McIntosh et al 2009)

Summary

Introduction

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone released into the blood stream in response to glucose and nutrients absorption from the intestine along with glucagon like polypeptide-1 (GLP-1) (Drucker 2006; McIntosh et al 2009) Both peptides are known to exert varied physiological actions on different body tissues with the pancreas being the major effector. These peptides interact with their respective G-protein-coupled receptors present on the cell surface and activate the adenylate cyclase signaling pathway. It brings about insulin secretion from the pancreas in a glucose-dependent manner and the name GIP. Techniques such as X-ray crystallography and NMR require proteins in mM

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Results