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).