Designing and constructing the encoding cassette of human epidermal growth factor for expression in barley seeds

Plants are very efficient in producing valuable pharmaceutical proteins. Human epidermal growth factor (hEGF) has numerous effects on various cellular systems, including wound healing, organogenesis, and so on. The present study was carried out with the aim of constructing monocotyledonous-specific vectors for hegf insertion and mass-production in a self-pollinated crop, barley, afterwards. This crop plant, with high protein yield and very low cross-pollination, is an ideal host for the production of epidermal growth factor.

The hegf sequence was optimized by COOL software, based on the codon preference of the host plant, and synthesized with a specific promoter, D-hordein. The encoding sequence of signal peptide targeting protein storage organelles in barley seeds was included at the beginning of the encoding area. The synthesized gene cassette was isolated from the cloning vector pUC57Hvorhegf by SacI and HindIII and cloned in pBI121. To insert the gene cassette of interest into pGH215, due to the lack of similar restriction sites in pUC57Hvorhegf and pGH215, the intermediate vector, pBI121Gus-12, was applied. After digesting pUC57Hvorhegf and pBI121Gus-12 by SacI and KpnI, the sequence of interest was incorporated into pBI121Gus-12. Finally, the recombinant pBI121Gus-12 and pGH215 were digested with KpnI and SalI, and the gene cassette was cloned at the right border of T-DNA, behind the NOS terminator.

Cloning accuracy and constructing pBI121Hvorhegf and pGH215Hvorhegf vectors bearing kanamycin and hygromycin, respectively, suitable for monocotyledons-crop transformation was confirmed by colony PCR, digestion pattern, and sequencing with forward and reverse primers.

The modified recombinant expression vector pGH215 with the gene of interest carrying the hygromycin gene cassette, giving a high level of ability to select transformed explants, can be used to transfer desired genes in monocotyledons and express those genes in protein storage organelles.