Biotechnol. Appl. Biochem. (2007) 48, 143–147 (Printed in Great Britain) doi:10.1042/BA20070004 143 Expression of human acidic fibroblast growth factor in Nicotiana benthamiana with a potato-virus-X-based binary vector Jingying Liu, Pengda Ma, Yan Sun, Meiying Yang, Liping Yang, Yuxi Li, Yanfang Wu, Xiaojuan Zhu 1 and Xingzhi Wang 1 Institute of Genetics and Cytology, Northeast Normal University, 5268 Renmin Street, Changchun 130024, People’s Republic of China The aFGF (acidic fibroblast growth factor) plays an important role in morphogenesis, angiogenesis and wound healing and is therefore of potential medical interest. A DNA fragment encoding haFGF (human aFGF) has been cloned into the PVX (potato virus X)-based binary vector (pgR107) and transiently expressed in Nicotiana benthamiana (a wild Australian tobacco) by agroinfection. Approx. 1 week after agroinfection, the recombinant haFGF accumulated in the agroinfected plants reached up to 1% of the total soluble protein. haFGF was then purified on heparin–Sepharose CL-6B. The purified haFGF could stimulate the growth of NIH 3T3 cells, suggesting that the recombinant haFGF expressed via PVX viral vector in N. benthamiana was active biologically. Introduction aFGF [acidic FGF (fibroblast growth factor)] is a member of a large FGF family consisting a group of structurally related polypeptides. To date, 23 FGFs have been isolated [1]. aFGF is a powerful mitogen involved in the stimul- ation of DNA synthesis and the proliferation of a wide variety of cell types, including fibroblasts, endothelial cells, smooth-muscle cells, myoblasts, chondrocytes and glial cells. It plays important roles in various stages of development and morphogenesis and also in angiogenesis and wound- healing processes [2–4]. For clinical applications, one needs to choose an optimal expression system to produce a large amount of aFGF in good quality and at low cost. Commercial protein production traditionally relies on microbial fermentation and mammalian cell lines. These systems, however, have disadvantages in terms of cost, scal- ability and safety that promote research effort into alter- natives. Despite industry inertia and conservatism, plants have turned out to be one of the most promising production platforms for the development of tomorrow’s biophar- maceuticals. Plants allow the cost-effective production of recombinant proteins on an agricultural scale, while eliminating risks of product contamination with endotoxins or human pathogens [5]. The first recombinant human gene to be expressed in plants was human growth hormone [6]. Since then, there has been much interest in the development of plants for the production of biopharmaceuticals [7]. Unlike the genes in stable transgenic plants, foreign genes expressed in plants utilizing virus vectors are not inherited to the next generation. Virus infection of a plant begins with the entry of infectious material into a cell, followed by amplification and accumulation of the virus, and then spreads throughout the entire plant. High expression level of the introduced genes and the rapid accumulation of the appropriate products make plant viruses well suited as transient expression vectors [8]. In recent years, the utilization of plant viruses as expression vectors to produce foreign protein has been studied widely. To date, many plant viruses have been designed as expression vectors, such as cowpea mosaic virus [9–12], tobacco mosaic virus [13–15] and PVX (potato virus X) [16–19]. In our study, we conducted experiments to use vector pgR107 for transient expression of the haFGF (human aFGF) in Nicotiana benthamiana (a wild Australian tobacco). pgR107 is a binary vector that was designed to combine the advantages of Agrobacterium tumefaciens-mediated transfection as well as virus infection [20]. We inserted the coding sequence of haFGF into the cloning sites of the viral binary vector to construct plasmid pgR107-haFGF. The haFGF recombinant protein was expressed in N. bentha- miana along with viral replication. Our goal was to achieve highly efficient plant haFGF production in a short time period. Key words: acidic fibroblast growth factor (aFGF), agroinfection, green fluorescent protein, mitogen, Nicotiana benthamiana (wild Australian tobacco), potato virus X. Abbreviations used: FGF, fibroblast growth factor; aFGF, acidic FGF; CP , coat protein; d.p.i., days post-agroinfection; haFGF, human aFGF; LB, Luria–Bertani; PVX, potato virus X; smGFP , soluble modified green fluorescent protein. 1 Correspondence may be addressed to either of these authors (email xingzhi@public.cc.jl.cn or zhuxj720@nenu.edu.cn). C  2007 Portland Press Ltd