Expression,Purification And Crystallization Of Rv2667 From Mycobacterium Tuberculosis

Tuberculosis is an infectious disease caused by bacteria in the Mycobacterium tuberculosis complex.Of these,the most common species to infect humans is M.tuberculosis.The TB bacillus is an extremely successful human pathogen,infecting two billion persons worldwide.Rv2667 is a hypothetical protein in mycobacterium tuberculosis.For now,there are no in vivo experiments for its functions.Rv2667 was predicted to contain no signal peptide which indicated that Rv2667 is not a secretive protein,and Rv2667 is functioning in cytoplasm,which conform with the AAA+ protease family.Rv2227 was predicted to possess no trans-membrane helixes,indicating that Rv2667 might not be a membrane protein.Moreover,Rv2667 was predicted to contain the clpA domain which belongs to the AAA+ proteases and might play roles in post-translation modification,protein degradation and molecular chaperone.The genome of mycobacterium tuberculosis H37Rv was extracted and used as template to amplify the Rv2667 ORF fragment.Rv2667 protein was expressed,purified and its crystalization condition was determined.The Rv2667 crystals exhibit the space group of P3112 with unit-cell parameters a = b = 48.07 A,c = 110.24 A,a=90°,p＝90°,γ=120°,and diffracted to a resolution of 2.1 A.The structure of hypothetical protein Rv2667 cannot be determined using molecular displacement method(MR),and the preparation of heavy atom derivatives are needed to provide phase analytical information.However,Rv2667 selenium protein derivative crystal needs to be further optimized.We also confirmed that Rv2667 exists in the form of hexamer.This study preliminarily confirmed that the hypothetical protein Rv2667 is functional,and offers a new strategy for drug design in tuberculosis.