Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis.
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%.
Ramalingam
B
B
Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V.R. Ramanathan Road, Chetput, Chennai 600 031, India.
Baulard
Alain R
AR
Locht
Camille
C
Narayanan
P R
PR
Raja
Alamelu
A
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014376:Tuberculosis / Q000175:diagnosis
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C430543:Rv3808c protein, Mycobacterium tuberculosis