טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShahar Anat
Subject3D Structure Determination of the Heat Shock Protein Cpn60.2
From Mycobacterium tuberculosis
DepartmentDepartment of Biotechnology
Supervisor Professor Noam Adir


Abstract

Heat shock proteins (HSP) are a large super-family of proteins which are highly conserved throughout evolution and are necessary for the correct folding of proteins inside the cell. Cpn60.2 from Mycobacterium tuberculosis (Mt) belongs to the HSP60 family which is also called Chaperonins. These proteins are involved in folding of a large number of proteins in an ATP dependent manner. In addition, Cpn60.2 is one of the most immunogenic of all Mt proteins, eliciting a significant immune response when whole cells are used in vaccination. Due to this high immunogenity, Cpn60.2 has a medical importance.

We have isolated Cpn60.2 by over expression of the cloned gene encoding for Cpn60.2 into pQE60 vector to enable metal chelate affinity purification. The recombinant protein was shown to protect E. coli cells from heat shock stress. Crystals of His-Cpn60.2 grow in 2-14 days and were improved by different methodologies. The crystallization conditions are 10% 2-propanol, 20% PEG 4K, 0.1M Hepes pH 7.5. Crystallographic analysis shows the crystals to be monoclinic (P21) with unit cell parameters of a=58.46Å, b=112.209Å, c=77.5Å, β=95.482° and containing a dimer in the asymmetric unit. We have collected a complete 2.75Å data on ESRF beam line ID14-1. The structure has been solved by the molecular replacement method using a lower resolution model recently published [1]. At present, the structure of the Cpn60.2 has been refined to R/Rfree factors of 21.5/29.2%.