The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 25 degrees. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn.HCl) or urea, show that functional bovine pancreatic ri...

Inclusion body refolding processes are poised to play a major role in the production of recombinant proteins. Improving renaturation yields by minimizing aggregation and reducing chemical costs are key to the industrial implementation of these processes. Recent developments include solubilization methods that do not rely on high denaturant concentrations and the use of high hydrostatic pressure...

The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and ...

Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles. Refolding of inclusion body proteins into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost in production of recombinant proteins from E. coli. With new information available on the structure and function of protein aggregates i...

Purpose: The refolding of recombinant human interferon α-2b is accompanied by low yield that could be due to high aggregation and multiplicity of steps in downstream processing. It is therefore essential to refold and purify the protein to simultaneously increase the production yield and reduce the required downstream steps. Methods: Inclusion bodies were dissolved in Tris-HCl buffer containing...

Background: The Hsp70 chaperone cycle mediates stress-denatured protein refolding. Results: We present the structure of a DnaK/GrpE complex containing the DnaK interdomain linker and substrate-binding domain. Conclusion: Interaction between the DnaK linker/lid regions and the GrpE N-terminal α-helix and disordered region are essential for function. Significance: The structure provides a framewo...

Background: Denileukin diftitox (trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering. Purity, stability, and structural property of the protein are critical factors for the scale-up production of this fusion protein. In this IM, loca...

Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concent...

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...