Codon-optimized recombinant expression assay for a monomeric 17kda basic fibroblast growth factor (bFGF) in transgenic alfalfa (Medicago sativa L.): blueprints towards human therapeutic proteins

LIU XIUMING; NAVEED AHMAD; ZHANG JIWEI; WANG ZHENGHAO; GUO YINGNAN; YAO NA; DONG YUANYUAN; WANG NAN; LI XIAOWEI; WANG FAWEI; LI HAIYAN; LI XIAOKUN

doi:10.30848/PJB2020-2(13)

LIU XIUMING Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China; College of Life Sciences, Jilin Agricultural University, Changchun 130118, China

NAVEED AHMAD Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China; College of Life Sciences, Jilin Agricultural University, Changchun 130118, China

ZHANG JIWEI Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

WANG ZHENGHAO Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

GUO YINGNAN Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China; College of Life Sciences, Jilin Agricultural University, Changchun 130118, China

YAO NA Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

DONG YUANYUAN Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

WANG NAN Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

LI XIAOWEI Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

WANG FAWEI Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

LI HAIYAN Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

LI XIAOKUN Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University,Changchun 130118,China

Basic fibroblast growth factor (bFGF) also called as FGF2/FGF-ß is an important growth factor and signal protein belongs to heparin binding growth factor family. The naturally occurring bFGF family is capable of stimulating a wide variety of biological activitiesand cellular responses including cell proliferation, angiogenesis, mitogenesis, embryonic development, cellular growth, wound repair process, oncogenic responses and neural development. The present work demonstrated the designing of a preferential codon optimizing strategy for bFGF recombinant protein by recruiting alfalfa as a suitable host bioreactor system. To validate our experimental design the over-expression vector p1390R carrying bFGF gene was efficiently transformed in to alfalfa using Agrobacterium mediated transformation. We have confirmed the successful integration of bFGF gene in alfalfa using various molecular techniques. In addition, the transcription regulation of bFGFin transgenic alfalfa was determined through RT-PCRthereby orchestrating gene activity.Subsequently, the recombinant protein of bFGF was separated on SDS-PAGE resulting in a 17kDa product. Moreover, the identification of specific bFGF single protein within a complex mixture of proteins was semi-quantitatively detected on western blot by estimating the size and color intensity of target protein. Altogether, our findings highlight anadvance molecular farming technique resulting in a controlled recombinant expression assay for a monomeric 17kDa bFGF protein in transgenic lines of alfalfa. The present work provides adequate information on the exploitation of plant host systems as bioreactors for essential recombinant therapeutic proteins such as vaccines, hormones, signaling molecules and antibodies with pharmacological potential in future researches.