Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein...

Since immobilized metal ion affinity chromatography (IMAC) was first introduced, several variants of this method and many other metal affinity-based techniques have been devised. IMAC quickly established itself as a highly reliable purification procedure, showing rapid expansion in the number of preparative and analytical applications while not remaining confined to protein separation. It was s...

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...

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea ...

Protein folding is subject to the effects of solvation environment. A variety of organic solvents are used as additives for in vitro refolding of denatured proteins. Examination of the solvent effects on protein folding could be of fundamental importance to understand the molecular interactions in determining protein structure. This article investigated the folding of α-helix and β-hairpin stru...

Whether turns play an active or passive role in protein folding remains a controversial issue at this juncture. Here we use a photolabile cage strategy in combination with laser-flash photolysis and photoacoustic calorimetry to study the effects of different turns on the kinetics of beta-hairpin refolding on a nanosecond time scale. This strategy opens up a temporal window to allow the observat...

effects of ?-cyclodextrin, ?cd, on refolding of lysozyme was investigated at ph 12 employing isothermal titration calorimetry (itc) at 300k in 30mm tris buffer solution. ?cd was employed as an anti-aggregation agent and the heats obtained for lysozyme+?cd interactions are reported and analyzed in terms of the extended solvation model. it was indicated that there are two sets of identical and no...

Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/...

Bacterially-produced recombinant prion protein (rPrP) is a frequently used model system for the study of the properties of wild-type and mutant prion proteins by biochemical and biophysical approaches. A range of approaches have been developed for the purification and refolding of untagged rPrP expressed as inclusion bodies in Escherichia coli, including refolding by dialysis and simultaneous o...