Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli

Abstract

Toward the future development of a defined subunit vaccine against leishmaniasis, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli. Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions. The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E. coli. When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E. coli. Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania. Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L. major GP63 expression clone as antigen.