a convenient method for solubilization and refolding recombinant proteins: an experience from recombinant mouse tgf-β1

Background: the production of recombinant proteins in escherichia coli (e.coli) is one of the most valuable achievements in biotechnology having many therapeutic and diagnostic applications; however, the aggregation and misfolding of proteins leading to the formation of insoluble inclusion bodies is a disruptive factor in this process. various solubilization and refolding methods could be used to improve recombinant protein conformation. in this study, we applied a dilution method with refolding buffer to produce a native form of soluble immature mouse transforming growth factor-beta 1 (tgf-β1). materials and methods: the tgf-β1 complementary dna (cdna) which encodes the protein without the signal peptide was cloned into the pet21-b (+) vector. the target protein was expressed by the transformation of e. coli bl21 cells with the plasmid. the resulting inclusion bodies were diluted in lysis buffer and solubilized in refolding buffer to make a protein with native structure. the protein quantification was performed by using bicinchoninic acid assay (bca). results: following polymerase chain reaction (pcr) of the recombinant plasmid with t7 primers, electrophoresis and sequencing of the amplified product indicated 100% target sequence identity with the murine tgf-β1 gene. finally, the protein solubility and immuno-reactivity were confirmed a 44 kda protein which conducted with the anti-tgf-β1-specific polyclonal antibody by western blot. the protein quantification with the bca method showed 30 μg/ml concentration. conclusion: the dilution method and refolding buffer used in this study could effectively convert aggregated immature mouse tgf-β1 to a soluble and immuno-reactive form.