Altering Trichomonas Vaginalis Codons Enhances Expression of The Trichomonad P270 Repeat Element in Escherichia Coli |Biomedgrid

The immunogenic protein of the Trichomonas vaginalis isolate NYH 286 called P270 (270-kDa) has 19 tandemly repeated elements (TRE), and each RE is 333-bp, which encodes for a protein of 11,770.90 daltons. Isolate NYH 286 trichomonads are infected with a dsRNA virus (TVV+), and TVV+ organisms undergo phenotypic variation between surface versus cytoplasmic placement of P270. These TVV+ trichomonads are heterogeneous with fluorescent and non-fluorescent parasites by indirect immunofluorescence using the monoclonal antibody (MAb) C20A3 as a probe. The p270 gene is single copy based on restriction enzyme analysis, and partial restriction using HindIII reveals a ladder pattern expected of TREs. Each TRE has the DREGRD epitope detected by MAb C20A3. As the cDNA encoding a protein of ~14-kDa within which is the ~11.8-kDa RE is poorly expressed by recombinant E. coli (rE.coli) the hypothesis was tested that the T. vaginalis codons for arginine (R) and glycine (G) translated by minor E. coli tRNA species decreased the synthesis of the RE. Therefore, the five codons of R109, R112, R116, G119, and G128 were altered with synonymous codons used by E. coli. The synthesis of the RE of the original plasmid (pORIG) expressed in rE. coli was then compared with rE. coli harboring the plasmid with less numbers of codons (called pLESS) used by the minor E. coli tRNAs. The control rE. coli with plasmid p21.b without insert, pORIG and pLESS all had similar growth kinetics, and bacterial lysates harvested at different times were evaluated by immunoblot for synthesis of the RE. The data show that the rE. coli with pORIG synthesized the RE only between 2-hours (h) and 8-h of growth. In contrast, rE. coli with pLESS permitted synthesis of the RE during the 30-h of bacterial growth. These data show for the first time the role of codon usage for expression of T. vaginalis proteins in rE. coli.