Construction of Mambalgin-۱ gene cassette for transient expression in apoplastic space

Mambalgine-۱ is a protein that acts by blocking ASICs channels in neurons as a potent analgesic such as morphine while having no adverse effects of morphine, such as addiction and respiratory distress. In this study, in order to produce inexpensive and scalable Mambalgin-۱ protein, recombinant PVX-Mambalgin-۱ viral vector was designed and optimized for transient expression in apoplastic space of Nicotiana benthamiana. To compensate for the lack of methionine at the beginning of Mambalgin-۱ and also secretion of this protein into apopalstic space Glucanase inhibitor protein ۲ (GIP۲) signal peptide was added to the N-terminal of Mambalgin-۱. The ۶xhis-tag was added to the C-terminal of Mambalgin-۱ for downstream processes and protein purification. Probability of signal peptide cleavage and secretory of Mambalgin-۱ assessed by SignalP.۵ server. The desired protein was reverse translated into nucleotide sequences based on N. benthamiana Codon Usage from Kazusa Database; then mRNA destabilizing sequences, polyadenylation signal and hidden stop codons were modified using Visual Gene Developer ۱.۹ and Mega۴ softwares. CAI and GC were kept as high as possible. ClaI/SalI restriction enzyme sites were added at the ۳’ and ۵’ end of the construct. The results showed that the GIP۲ signal peptide would most likely (۰.۹۶) be cleaved from Mambalgin-۱ and Mambalgin-۱ expression will be highly secretory (۰.۹۹۸) into the apopalstic space after expression. After optimization, cloning and transfection of Agrobacterium, the existence and transformation of recombinant PVX-Mambalgin-۱ vector was confirmed by colony PCR.Mambalgine-۱ is a protein that acts by blocking ASICs channels in neurons as a potent analgesic such as morphine while having no adverse effects of morphine, such as addiction and respiratory distress. In this study, in order to produce inexpensive and scalable Mambalgin-۱ protein, recombinant PVX-Mambalgin-۱ viral vector was designed and optimized for transient expression in apoplastic space of Nicotiana benthamiana. To compensate for the lack of methionine at the beginning of Mambalgin-۱ and also secretion of this protein into apopalstic space Glucanase inhibitor protein ۲ (GIP۲) signal peptide was added to the N-terminal of Mambalgin-۱. The ۶xhis-tag was added to the C-terminal of Mambalgin-۱ for downstream processes and protein purification. Probability of signal peptide cleavage and secretory of Mambalgin-۱ assessed by SignalP.۵ server. The desired protein was reverse translated into nucleotide sequences based on N. benthamiana Codon Usage from Kazusa Database; then mRNA destabilizing sequences, polyadenylation signal and hidden stop codons were modified using Visual Gene Developer ۱.۹ and Mega۴ softwares. CAI and GC were kept as high as possible. ClaI/SalI restriction enzyme sites were added at the ۳’ and ۵’ end of the construct. The results showed that the GIP۲ signal peptide would most likely (۰.۹۶) be cleaved from Mambalgin-۱ and Mambalgin-۱ expression will be highly secretory (۰.۹۹۸) into the apopalstic space after expression. After optimization, cloning and transfection of Agrobacterium, the existence and transformation of recombinant PVX-Mambalgin-۱ vector was confirmed by colony PCR.