Production of a recombinant human basic fibroblast growth factor with a collagen binding domain

Abstract

Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [3H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [3H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.

Construction of multifunctional proteins for tissue engineering: Epidermal growth factor with collagen binding and cell adhesive activities

“…To do so, a collagen binding domain (CBD) was fused to the previously described ERE-EGF (Elloumi et al, 2006). CBD can have different origins and its fusion to growth factors was reported (Kato et al, 2007;Nimni, 1997;Takagi et al, 1991Takagi et al, , 1992Hoyer, 1981;Andrades et al, 2001). In the present study, we used a fibronectin-derived CBD (fnCBD) (Ishikawa et al, 2001).…”

The small leucine‐rich proteoglycan biglycan modulates BMP‐4‐induced osteoblast differentiation

“…In contrast, little is known about the role of the ECM on osteogenic cells during the process of maturation. There are several reports that type I and II collagens and the small leucine-rich proteoglycans, including biglycan (bgn) and decorin (dcn), can bind growth factors (7)(8)(9). Based on this evidence, a theory has arisen that the ECM may play an important role as a reservoir for balancing the activities of growth factors to control osteoblast differentiation.…”

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

“…In animal tissues the content of natural bFGF is very limited and its puri cation in quantities that meet the market demand is almost impossible (Gasparian et al, 2009). As a result, during past three decades, considerable efforts have been made for the expression of recombinant bFGF in different organisms including Escherichia coli (Andrades et al, 2001;Gasparian et al, 2009;Jia et al, 2015;Ke et al, 1992;Kwong et al, 2016;Rassouli et al, 2013;Song et al, 2013;Squires et al, 1988;Ping et al 2005;Imsoonthornruksa et al 2015;Soleyman et al 2016), Saccharomyces cerevisiae (Barr et al, 1988), Bacillus subtilis (Kwong et al, 2013), Pichia pastoris (Mu et al, 2008), Bombyx mori L. (silkworm) , rice and soybean (Glycine max) (Ding et al, 2006) seeds, and tobacco (Nicotiana tabacum) chloroplasts (Wang et al, 2015). Currently, the commercial bFGF is manufactured in recombinant form mainly in The high expression levels of production of recombinant human bFGF in E. coli is accompanied by problems including misfolding and the formation of insoluble aggregates known as inclusion bodies, which renders the recombinant proteins to be biologically inactive.…”