title: Engineering, expression and renaturation of targeted TGF-beta fusion proteins.
abstract: This study reports the expression, purification, and renaturation of biologically active Transforming Growth Factor-beta 1 (TGF-beta 1) fusion proteins from Escherichia coli (E. coli). A prokaryotic expression vector was engineered to produce tripartite fusion proteins consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen binding domain, and (iii) a cDNA sequence encoding the active fragment of human TGF-beta 1. The expressed fusion proteins TGF-B1-F1 and TGF-B1-F2, located in inclusion bodies, were solubilized with 8 M urea and renatured using a glutathione redox-coupled system and protracted dialysis under several experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion proteins on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to inhibit mink lung (Mv1Lu) cell proliferation and/or to stimulate proliferation of NIH-3T3 mouse fibroblasts, where purified human platelet TGF-beta 1 served as a positive control. Purified TGF-B1-F1 and TGF-B1-F2 (collagen-binding) constructs exhibited anti-proliferative activities comparable to purified platelet TGF-beta 1, but at lower specific activities. Binding of the renatured TGF-B1-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of 3H-collagen to the TGF-B1-F2 protein immobilized on a Ni-NTA column. The TGF-B1-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active TGF-beta 1 fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, these results support the concept that auxiliary domains may be used to modulate and/or target TGF-beta 1 for specific applications.
authors.affiliation: Research Institute Childrens Hospital Los Angeles, University of Southern California School of Medicine 90027, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Tuan
authors.forename: D T
authors.initials: DT
authors.lastname: Cheung
authors.forename: L T
authors.initials: LT
authors.lastname: Wu
authors.forename: A
authors.initials: A
authors.lastname: Yee
authors.forename: S
authors.initials: S
authors.lastname: Gabriel
authors.forename: B
authors.initials: B
authors.lastname: Han
authors.forename: L
authors.initials: L
authors.lastname: Morton
authors.forename: M E
authors.initials: ME
authors.lastname: Nimni
authors.forename: F L
authors.initials: FL
authors.lastname: Hall
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D051379:Mice; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D003094:Collagen