Optimization of human basic Fibroblastic Growth Factor coding sequence for expression in rice (Oryza sativa L.) and cloning under ramy3D gene regulating sequences in the binary vector pCAMBIA1304

Human basic Fibroblastic Growth Factor (hbFGF) is a valuable protein with a wide variety of clinical and research applications. Currently, recombinant production of this growth factor in conventional platforms of microbial and insect cells is accompanied by several problems causing high cost production that limited its applications. Rice cell suspension culture is an efficient, safe and low cost platform for recombinant protein production. In this study we prepared a suitable construct for high level expression of an optimized version of bFGF coding sequence under control of ramy3D gene expression sequences in rice cell suspension culture. The codon usage and GC content of bFGF coding sequence was optimized for expression in rice and then synthesized. Following optimization, the Codon Adaptation Index and GC content were upgraded from 0.76 to 0.82 and from 52.65% to 55.45%, respectively. The ramy3D regulating sequences were amplified from genomic DNA in two separate fragments and then cloned. The ramy3D regulating fragments were assembled in the binary vector pCAMBIA1304. Then the bFGF optimized CDS was inserted in the construct between signal peptide CDS and 3′UTR. Ramy3D expression system has several advantages including high level expression of transgene, inducibility and simple protein purification through protein secretion into the medium.