It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in...

Background: Tuberculosis as a global health problem requires special attention in the contexts of prevention and control. Subunit vaccines are promising strategies to protect burdens of tuberculosis via increasing the BCG protection. The present study aimed to design a vaccine study by means of high-throughput expression and correct refolding of recombinant protein PPE17. Methods: We aimed to c...

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification ...

L-arginine (Arg) is a widely used additive for suppressing protein aggregation during refolding. Systematic screening of Arg analogs provides superior additives that enhance the refolding yield more effectively than Arg. The refolding yield of hen egg lysozyme in the presence of 500 mM L-argininamide (ArgAd) increases 1.7-fold higher than Arg. Thermal unfolding experiments indicate that ArgAd h...

Three foldases--the apical domain of GroEL (mini-chaperone) and two oxidoreductases (DsbA and DsbC) from Escherichia coli--were studied in refolding a protein with immunoglobulin fold (immunoglobulin-folded protein) that had been produced as inclusion bodies in E.coli. The foldases promoted the refolding of single-chain antibody fragments from denaturant-solubilized and reduced inclusion bodies...

The impact of the quenched force on protein folding pathways and free energy landscape was studied in detail. Using the coarse-grain Go model, we have obtained the low, middle, and high force regimes for protein refolding under the quenched force. The folding pathways in the low force regime coincide with the thermal ones. A clear switch from thermal folding pathways to force-driven pathways in...

Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inclusion bodies, (2) change in conformation and flexibility or solubility of pro...

The Anfinsen hypothesis, the demonstration of which led to the Nobel prize in Chemistry, posits that all information required to determine a proteins' three dimensional structure is contained within its amino acid sequence. This suggests that it should be possible, in theory, to fold any protein in vitro. In practice, however, protein production by refolding is challenging because suitable refo...

This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression insoluble protein in Escherichia coli followed by in-column chemical refolding. allowed detailed comparisons with perdeuterated produced yeast Pichia pastoris , as well unlabelled lysozyme. Both variants exhibit reduced thermal stability enzymatic activity comparison hydroge...