3.1 Creation and characterization of transgenic wheat seeds expressing recombinant TM-1.
Lower expression of plant made biopharmaceutical is a great concern and hurdle for its commercialization. Several efforts including enhanced promoters, regulatory elements, protein targeting, utilization of various plants and their tissues and codon optimizing improve overall yield. It has been known that mRNA stability and translational efficiency are enhanced by codon optimization due to idealized tRNA usage preference of recombinant TM-1 biosynthesis in host cells [20]. Therefore, coding sequence of TM-1 was optimized during which 88 out of 262 codons in native sequence were replaced, resulting in an elevated GC% from 31.93% to 46.44% (supplementary Fig. S1). The rice seed-specific promoter GluB-4 was included in construct as it can drive and target recombinant protein in rice endosperm with the highest translational activity among other 14 rice seed-specific promoters [17] (Fig. 1A). The ER retention signal peptide KDEL linked to 3’ end of TM-1 coding sequence has been shown to dramatically increase recombinant protein expression in rice endosperm [21]. CTB fused immediately upstream of TM-1 allows transport of TM-1 through gastrointestinal epithelia to circulation system by binding to a mucosal membrane receptor ganglioside GM1 [20]. Furin cleavage site can be digested by furin, a ubiquitous protease in human cells, enabling release of TM-1 after transmucosal transfer carried by CTB [22].
Three independent transgenic wheat lines were generated with particle bombardment and its subsequent regeneration and selection, and the line with highest expression was selected for further analysis. To verify the insertion of CTB-TM-1 construct in wheat genome, PCR with construct-specific primers was performed using genomic DNA from wheat T1 transgenic endosperm (Fig. 1B). The presence of CTB-TM-1 construct was detected in all three lines (Fig. 1B). The Western blot further confirmed the presence of recombinant TM-1 in wheat seeds from all lines with mean expression level of 0.84±0.046, 1.03±0.068, and 0.92±0.096 mg/g dry weight (DW) respectively and correct size as expected (~41 kDa) (Fig. 1C and 1D). To evaluate the proper formation of CTB pentamer that binds GM1 receptor, a GM1 ELISA assay was carried out with crude extract of wheat seeds expressing recombinant CTB-TM-1. The results showed CTB-TM-1 correctly bond to GM1 in vitro when compared to CTB standard (Fig. 1E). In addition, unless otherwise indicated, the T1 seeds of line TM-1-2 was used for all downstream analysis and processes due to its highest TM-1 expression level and prominent affinity to GM1 receptor.
The transgenic nature of wheat plants transformed with CTB-TM-1 cassette by biolistics were tested on MS ½ media containing antibiotic as selection marker. All the embryo excised from PCR confirmed transgenic T0 plants showed efficient regeneration as compared to wild type embryos as negative control. Total of 25 embryos excised from transgenic wheat line were confirmed by transgene integration and showed regeneration (Fig. 2).