Microbial host systems remain the most efficient and cost-effective chassis for biotherapeutics production. Escherichia coli is often preferred due to ease of cloning, scale-up, high product yields, importantly, cultivation. E. experience difficulties in producing biologically active therapeutics such as Fab fragments, which require protein folding subsequent three-dimensional structure develop...

Oxidative refolding of the denatured/reduced lysozyme was examined in the presence of small unilamellar vesicles (SUVs) essentially composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). SUVs facilitated the recovery of the enzymatic activity of lysozyme like molecular chaperones through the interaction with the refolding intermediate of lysozyme. The highest reactivation yield (8...

Prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206-Cys210, and Cys250-Cys283. We have demonstrated that Cys250-Cys283 is indispensable for correct refolding of prochymosin, whereas Cys45-Cys50 is dispensable but has some contribution to the stability and substrate specificity of the enzyme. Here, we report the results about the functions of Cys206-Cys210 by site-directed...

Refolding of the very-fast-folding unfolded species (Uvf) of disulfide-intact bovine pancreatic ribonuclease A has been monitored by circular dichroism (CD) at 222 and 275 nm at 0.9 or 2.6 M guanidine hydrochloride, pH 7.0, and 5 degrees C. The refolding of Uvf represents a purely conformational folding process which is not complicated by cis-trans proline isomerization. The data indicate that ...

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agen...

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 ...

Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts. We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10. In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent Kd of 0.9 nM and a second molecule of hsp10 binds with a Kd of 24 nM. In the presence of ATP, the purified yeast chaperonins mediate...

Attachment invasion locus (Ail) protein of Yersinia pestis is a crucial outer membrane protein for host invasion and determines bacterial survival within the host. Despite its importance in pathogenicity, surprisingly little is known on Ail biophysical properties. We investigate the contribution of micelle concentrations and interface tryptophans on the Ail β-barrel refolding and unfolding proc...

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubil...