Cloning, expression, and puriWcation of the 27kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis

Ramalingam, B and Baulard, A R and Locht, C and Narayanan, P R and Raja, Alamelu (2004) Cloning, expression, and puriWcation of the 27kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis. Protein Expression and Purification , 36 (1). pp. 53-60. ISSN Print: 1046-5928; Online: 1096-027

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Abstract

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 puriWcation of a 27kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR ampliWed using primers that contain speciWc restriction sites. The ampliWed 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 puriWed 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-Xight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The puriWed 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 speci- Wcity of 95%.

Item Type:	Article
Uncontrolled Keywords:	Pulmonary tuberculosis; 27 kDa antigen; Cloning; Over-expression; HPLC; ELISA
Subjects:	Tuberculosis > Laboratory Research > Immunological
Divisions:	Basic Science Research > Immunology
Depositing User:	Dr. Rathinasabapati R
Date Deposited:	21 Nov 2013 08:10
Last Modified:	09 Mar 2016 10:31
URI:	http://eprints.nirt.res.in/id/eprint/660

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