The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C. Changes in the intrinsic protein fluorescence on refolding are characterized by four phases. Based on changes in the amplitudes of these phases, as a consequence of quenching of th...

Background: The production of recombinant proteins in Escherichia coli is one of the most valuable achievements in biotechnology, with many therapeutic and diagnostic applications; however, the aggregation and misfolding of proteins that result in the formation of insoluble inclusion bodies is a disruptive factor in this process. Various solubilization and refolding methods can be used to impro...

Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length protei...

Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding...

Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by opt...

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of beta-galactosidase and luciferase were examined at low substrate concentration and low...

Chromatographic columns packed with commercially available hydrophobic interaction chromatography (HIC) media were found to be able to suppress aggregation and nevertheless had a tendency to promote the structural misfolding resulting in higher soluble protein recovery and lower specific activity than that by dilution when they were used to refold lysozyme, a model protein. Moreover, this misfo...

BACKGROUND Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain di...

Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes i...

Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine which is widely used for treating neutropenia in humans. Evaluation of alternative to expensive components of redox buffer (reduced and oxidized glutathione) is an important step in reducing the cost of production of human biotherapeutic proteins. In the present study, refolding of recombinant human G-CSF expressed as i...