Biochemical Properties of Recombinant Tyla Protein From Mycobacterium Tuberculosis

Abstract

Mycobacterium tuberculosis (M.tb), the causative agent of the disease tuberculosis, is an newlineancient pathogen infects one third of the world s population claiming 1.5 million lives newlineannually- a WHO report. TlyA is a virulence factor that is evolutionarily conserved in many newlinegram-positive bacteria. newlineIn the present study, cloning of Mycobacterium tuberculosis Tly A in pET-Duet vector newlinebetween EcoRI and KpnI and the clone was confirmed by sequencing . Tly A was confirmed newlineby Western blot analysis using and#945;-his antibodies. Protein was expressed in BL21 cells and newlineprotein expression was optimized using different temperature and IPTG concentrations. newlineOptimum expression of protein observed at 300C and at 500µM IPTG concentrations. Tly A newlinewas purified by two chromatography techniques Ni-NTA chromatography followed by gel newlinefiltration. Purified protein showed monomer in gel filtration and MALDI-TOF. Purified Tly A newlineshowed haemolytic activity indicating that the protein is active. Nucleotide sequence analysis newlineof cloned M.tb TlyA encodes for 268 amino acids with a molecular mass of and#8764;29.9 kDa newlineincluding 16 additional amino acids, molecular weight of E59Q Tly A is consistent with the newlineTly A native protein. The native protein is reported to have autocatalytic activity, whereas newlineE59Q did not show any autocatalytic activity. Secondary structural studies using the CD newlinespectrum of both nativeand E59Q showed the pattern of well folded protein with a mixed and#945;/and#946; newlinestructure and E59Q mutant protein is quite stable without any degradation after purification. newlineMass spectrometry studies indicated that protein is ~30 kDa which is evident from the newlineprevious reports. M.tb-Tly A NTD plays an important role and is dispensable for interaction newlinewith the substrate, M.tb-TlyA NTD expressed with the maltose binding protein (MBP) tag. CD newlinestudies showed disordered structure of the TlyA NTD is observed at 0.4 mg/ml, whereas newlineincreasing concentrations increased helical content and showed ordered structure. A high newlineresolution X-ray crystal structure of the TlyA ful