Effect of signal peptide type on recombinant anti-HIV griffithsin protein accumulation in transgenic tobacco plants

Atend of 2010 year, about 34 million people living with HIV. With current therapy AIDS is still not curable. Griffithsin (GRFT) is a potent anti-HIV protein that binds to HIV and inhibits cell to cell transmission of virus. In this study, first GRFT gene sequence was optimized. Then pBIgR-KD and pBIgR-ZR vector containing GRFT gene plus N termination of Zera SP ZERA and endoplasmic reticulum retention signal peptide (SEKDEL) was constructed. To obtaining transformants, young leaf explants of Nicotiana tobacco cv. Samsun were transformed with agrobacterium LBA4404 strain containing pBIgR-KD and and pBIgR-ZR. Formed shoots on MS medium containing 1 mg/l BA and 0/1 mg/l NAA and 100 mg/l kanamycin were transferred to root inducing medium (½ MS hormone free) containing 50 mg/l Kn. PCR using GRFT and 35S primers and sothern blot analysis showed succefull integration of GRFT transgene in tobacco genome. RT-PCR and real time PCR showed expression of GRFT gene at transcriptome level with high differences of expression transgenic lines. A band with corresponding size (27 KD for dimer GRFT protein) was detected based on SDS-PAGE analysis. Western blot analysis with anti-GRFT antibody confirmed production of GRFT protein as monomer (13 KD) and dimer (27 KD). Based on ELISA data, targeting to protein bodies using Zera signal peptide resulted in higher amount of recombinant GRFT in comparison to ER targeting via KDEL signal peptide. Based on our results high level of recombinant protein expression is achievable by optimizing of gene codon, selection of suitable host plant and application of suitable promoter and signal peptides.