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A Journal on Biotechnology and Molecular Biology
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,246
Minerva Biotecnologica 1999 March;11(1):17-22
Production of a recombinant Treponema pallidum diagnostic antigen in E. coli using three different expression strategies
Del C. Dominguez M. 1, Acevedo B. 1, Béniex J. 1, Miranda A. 1, Canaan L. 2, Galivondo J. V. 1
1 Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology, La Habana, Cuba;
*Division of Chemistry-Physics, Center for Genetic Engineering and Biotechnology, La Habana, Cuba
Background. Among treponemal antigens, the 42 kDa TmpA membrane protein has been reported as potentially useful in the development of ELISA diagnostic tests for primary, secondary, and latent syphilis. In our work we tested cloning methods and expression strategies for the TmpA antigen and compared the TmpA fusion proteins with respect to their performance in a diagnostic ELISA assay.
Methods. In this paper we report the isolation of the full TmpA gene from patient material, using the polymerase chain reaction. The gene was inserted in three expression vectors controlled by tryptophan promoter. Recombinant TmpA variants were provided with multihistidine tags, and purified in one single step using immobilized metal affinity chromatography. We compared the three TmpA fusion proteins with respect to their performance in a diagnostic ELISA assay.
Results. The recombinant TmpA variant in E. coli as a soluble cytoplasm fusion protein was highly expressed. TmpA expression was also directed to the periplasm via an ompA signal peptide, or produced insoluble and associated with the bacterial inner membrane after an N-terminus addition of a 24 aminoacid domain. These antigens showed adequate performance in ELISA when tested with a panel of human sera, versus VDRL.
Conclusions. These results indicate that the recombinant TmpA is a good option for the diagnosis of syphilis by ELISA and that the inclusion of different protein fragments into the recombinant antigen will not necessarily affect the antigenicity of a protein to be used in diagnostic procedures.