Health & Environmental Research Online (HERO)


Print Feedback Export to File
1851826 
Journal Article 
Cloning, expression, and purification of the 27kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis 
Ramalingam, B; Baulard, AR; Locht, C; Narayanan, PR; Raja, A 
2004 
1 
Protein Expression and Purification
ISSN: 1046-5928 
36 
1 
53-60 
English 
15177284 
WOS:000222037200007 
A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%. 
pulmonary tuberculosis; 27 kDa antigen; cloning; over-expression; HPLC; ELISA