Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

Abstract

Human basic fibroblastic growth factor (hbFGF), possesses a wide range of highly important biological functions, including cell proliferation, protein synthesis and metabolism. This study is the first investigation to transfer a partly home-made hbFGF gene cassette into rice cells. The human bFGF coding sequence was optimized for expression in rice in order to improve its in vitro production. Following synthesis, the expression cassette was constructed by fusing the PCR- amplified Ram3D promoter and terminator sequences in two separate steps to the optimized hbFGF CDS. Then, the cassette was transferred into the pCAMBIA1304 shuttle vector. This vector also contained a signal peptide for the secretion of the recombinant protein into the culture medium. Rice (Oryza sativa cv. Kanto 51) seeds were cultured on the 2N6PG medium composing of the N6 basal medium supplemented with 2 mg/L 2,4-D for callus induction. Callus transformation was performed by employing Agrobacterium tumefaciens strain LBA 4404. The integration of bFGF gene into the chromosomes of transgenic calli was verified by genomic DNA PCR. A transformed callus line expressing the highest level of bFGF (38.1 mg/kg FW) was recognized by ELISA and selected for the establishment of cell suspension culture. The developed transgenic rice cell culture was able to express and secrete the recombinant bFGF into the culture medium on day 3 after incubation of cells in the sucrose-free medium. The SDS-PAGE and Western blot analyses detected the bFGF in the culture medium with a molecular weight of ~17 kDa under reducing conditions. The purification of bFGF was performed using HiTrap Heparin HP column. Interestingly, the activity assay indicated that the rice derived bFGF stimulated the proliferation of NIH/3T3 cells similar to the commercial bFGF.