Heterologous production of recombinant anti-HIV microbicide griffithsin in transgenic lettuce and tobacco lines

Abstract

We aimed to evaluate the possibility the nuclear transformation of lettuce and tobacco to produce recombinant anti-HIV microbicide griffithsin under impact of Zera signal peptide. For this purpose, the codon optimized GRFT fused with KDEL retention signal was used with and without the Zera (γ-zein ER-accumulating domain) signal peptide. Integration of GRFT into the nuclear genome of lettuce and tobacco transgenic lines was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Subsequent reverse transcription-quantitative PCR (RT-qPCR) experiments showed highly divergent GRFT expression patterns, inherent to the applied transformation procedure. The recombinant GRFT was successfully detected by means of western blot and quantified by ELIZA. According to ELIZA results, fusion of GRFT with Zera signal peptide resulted in higher accumulation of the recombinant protein in both species once compared with transgenic line without signal peptide. Lettuce showed higher transgene transcripts and accumulated more the recombinant protein of interest (up to 8.942 µg/100 mg) than tobacco. Both lettuce- and tobacco-derived GRFT (GRFTL and GRFTT, respectively) captured gp120 in a way comparable to E. coli expressed GRFT (GRFTE). Our results suggest that lettuce as a leafy vegetable crop and tobacco as a model plant in transgenic research studies can be used as suitable candidate hosts for the production of recombinant GRFT, augmented by recruitment of plant optimized codon compositions and suitable signal peptide.