MS Thesis Defense of Ms Safia Niaz
Title: Expression and purification of Drosophila polycomb protein
Supervisor: Muhammad Tariq
Venue: SSE smart lab 4th floor
Date: Monday, May 22nd, 2017
Time: 03:00 pm
In Drosophila, during the embryo development, homeotic (Hox) genes play an important role in specifying different segments in the anterior-posterior axis of the body. Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for regulating the expression of Hox genes antagonistically. PcG is divided into three groups, Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2) and Pho-repressive complex (Pho-RC) which are involved in maintaining the repressed state of genes. PRC2 is recruited to the PRE region of the respective gene via Pho-RC and adds a trimethyl mark on H3K27. This H3K27me3 is recognized by chromodomain of Polycomb (Pc) protein which is the member of PRC1. Pc, with the help of its C-terminus, recruits other members of PRC1 complex i.e. Polyhomeotic (Ph), Posterior sex comb (Psc) and Sex comb extra (Sce) to maintain the repressed expression of the genes by inhibiting the elongation of RNA polymerase II or by causing the compaction of chromatin. In 1992, Franke et al observed that deletion of chromodomain abolished the chromosomal binding of Pc (Messmer, Franke et al. 1992) while truncations in its C-term caused redistribution of Ph protein and lethality of Drosophila embryos. Pc protein plays important role in the repression of more than 100 genes but the exact mechanism of silencing is not fully understood. In this project, we have expressed the full length Pc protein in Pichia pastorisvia secretory pathway and purified via FPLC techniques in order to better understand the silencing mechanism by employing structural techniques.
Pichia pastoris is methylotrophic yeast that metabolizes methanol as its sole carbon source.Pichia shows many features of eukaryotic expression systems like protein processing, protein folding and particularly post translational modifications like methylation and acetylation. It grows fast, is easy to manipulate and less expensive as compared to eukaryotic expression systems. PC gene is cloned into pPIC9 vector which is transformed in two Pichia pastoris strains, GS115 and KM71. Pc protein is expressed in successfully transformed cells and purified via FPLC techniques after optimization of washing and elution conditions. The purified protein is confirmed by SDS-PAGE and western blot analysis.