Refolding of a recombinant collagen-targeted TGF-beta2 fusion protein expressed in Escherichia coli.
In this study, a tripartite transforming growth factor-beta (TGF-beta2) fusion protein bearing an N-terminal purification tag and an auxiliary collagen binding decapeptide has been constructed and expressed at high levels in Escherichia coli. The resulting recombinant protein accumulates in an insoluble and biologically inactive inclusion-body complex. The insoluble protein was solubilized in guanidine hydrochloride and a Ni-chelating affinity column was utilized to isolate the 13.5-kDa TGF-beta2 fusion protein, which was then refolded into its native conformation under controlled redox conditions. The formation of native homodimers was monitored by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels and the bioactivity determined by a quantitative TGF-beta assay system using mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter/luciferase reporter plasmid. To optimize yields, renaturation conditions including denaturants, limiting protein concentrations, redox ratios, dialysis conditions, and refolding kinetics were studied and monitored by bioactivity. These studies demonstrate that recombinant TGF-beta2 fusion proteins can be produced in E. coli and renatured into biologically active homodimers. Furthermore, they confirm that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen. Taken together, these studies advance the technology necessary to generate large quantities of targeted TGF-beta fusion proteins for specific biomedical applications.
Han
B
B
School of Medicine, University of Southern California, Los Angeles, California 90027, USA.
Hall
F L
FL
Nimni
M E
ME
D001681:Biological Assay; D003094:Collagen / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D003094:Collagen