TY - JOUR T1 - Gene cloning and construction of prokaryotic and plant expression vectors of RICIN-A-Chain/PAP-S1 fusion protein and its inhibition of protein synthesis JF - bioRxiv DO - 10.1101/046060 SP - 046060 AU - Yasser S. Hassan AU - Sherry L. Ogg Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/03/31/046060.abstract N2 - Pokeweed antiviral protein (PAP) is a single-chain ribosome-inactivating protein that exists in several forms isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). In this study, PAP-S1, one of the two known isoforms found in seeds, was isolated and PCR amplified using primers based on the known mRNA of PAP-S2, the other known form found in seeds. The complete cDNA encoding PAP-S1 was determined here for the first time. PAP-S1 is a potent antiviral protein with many potential clinical applications. However, it was found to be dosage dependent with observed side effects at high dosage. In this study, we report the production of a recombinant antiviral peptide-fusion protein between Ricin A-chain and PAP-S1. The peptide-fusion recombinant proteins Ricin-A-Chain/PAP-S1 and PAP-S1/Ricin-A-Chain were generated by joining the Nterminus of PAP-S1 to the C-terminus of Ricin A-chain and the C-terminus of PAP-S1 to the N-terminus of Ricin A-chain respectively, and were expressed in an Escherichia coli cell free expression systems. The peptide-fusion recombinant protein Ricin-A-Chain/PAP-S1 (F2) was found to be more active than the PAPS1/Ricin-A-chain (F1) and similar to PAP-S1 in a cell free prokaryotic environment, and both showed much stronger activity in a cell free eukaryotic environment. The DNA sequence of the complete cDNA of PAP-S1 and of the peptide-fusion protein Ricin-A-Chain/PAP-S1 with the PAP-S1 signal peptide at the N-terminus of Ricin Achain were inserted in plant destination binary vectors for A. tumefaciens mediated transformation. It is the authors’ opinion that additional research should be done in order to determine both cytotoxicity and selectivity of fusion protein F2 compared to PAP-S1, as it could be a viable, more potent and less cytotoxic alternative to PAPS1 alone at high dosage, for both agricultural and therapeutic applications. ER -