Production and Purification of the p16INK4a:  A Naked Mole-Rat’s Resistance Protein to Cancer

João Alberto Pacheco Marques de Vasconcelos e

Key information

Authors:

João Alberto Pacheco Marques de Vasconcelos e (João Alberto Pacheco Marques de Vasconcelos e Sá)

Supervisors:

Alexander Dikiy (Alexander Dikiy); Maria Ângela Cabral Garcia Taipa Meneses de Oliveira (Maria Ângela Cabral Garcia Taipa Meneses de Oliveira)

Published in

11/30/2012

Abstract

The aim of the present work was the purification of p16INK4a protein from naked mole-rat (nmr) with the objective of performing NMR experiments. These experiments would serve to obtain the necessary information for determination of its tridimensional structure. Naked mole-rat (Heterocephalus glaber) is a remarkable mammal in which spontaneous cancer has never been observed. Experiments showed that the protein p16INK4a, encoded in the INK4a/ARF locus, plays an important role in tumor suppression by supporting early contact inhibition, a mechanism absent in human cells. Moreover, this locus uniquely changed in nmr, generates shorter forms of both protein products synthesized from this locus. Thus, analysis of the structure and function of p16INK4a may provide an explanation for the role of this protein in cancer. Due to the recent completion of the nmr’s genome project, the proposed work is original and innovative. In a first phase, the plasmid pET28B in E. coli BL21 (DE3) was used, which produced p16 with a histidine tag was used, and in a second phase the plasmid pGEX-4T1 in E. coli BL21 (DE3) which produced p16 with a GST tag was used. In both expression systems pure concentrated p16 was not achieved, due to precipitation of the protein which was not overcome, despite several conditions have been tried. Thus, it seems that the precipitation is due intrinsically to p16 and possible alternatives are to use more soluble fusion partners, co-expression with chaperones or even perform NMR or x-ray crystallography experiments with the protein still fused to GST.