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Microbial Cell Factories Mar 2015Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from... (Review)
Review
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 optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.
Topics: Escherichia coli; Inclusion Bodies; Models, Molecular; Protein Denaturation; Protein Folding; Protein Refolding; Protein Unfolding; Recombinant Proteins; Solubility
PubMed: 25889252
DOI: 10.1186/s12934-015-0222-8 -
Biomacromolecules Sep 2022We have reported that ureido polymers exhibit upper critical solution temperature (UCST)-type phase behavior in solution, which is the opposite of lower critical...
We have reported that ureido polymers exhibit upper critical solution temperature (UCST)-type phase behavior in solution, which is the opposite of lower critical solution temperature (LCST)-type behavior. Furthermore, UCST-type ureido polymers undergo liquid-liquid phase separation (LLPS) upon cooling rather than the liquid-solid phase transition of the typical LCST-type polymers. In this study, ureido polymers with hydrophobic groups were prepared to evaluate the effects of cooling-induced LLPS of UCST-type polymers on refolding of proteins. When protein was heated with a ureido polymer functionalized with undecyl groups, aggregation of the protein was prevented. Subsequent cooling incubation resulted in the spontaneous release of the protein from the polymer. The released protein had enzymatic activity, suggesting that the protein refolded properly. Interestingly, efficient refolding was observed when the solution of the UCST-type ureido polymer and protein was incubated at around the phase separation temperature of the polymer, implying that cooling-induced LLPS of the polymer enhanced the release of the protein. Additionally, by centrifugation at 4 °C, the refolded protein was readily separated from the ureido polymers, which precipitated upon cooling.
Topics: Hydrophobic and Hydrophilic Interactions; Phase Transition; Polymers; Protein Refolding; Proteins; Temperature
PubMed: 36030420
DOI: 10.1021/acs.biomac.2c00694 -
Biotechnology Journal Jan 2013In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science... (Review)
Review
In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-based methods that contribute to the emergence of a universal refolding method.
Topics: Biotechnology; Models, Molecular; Protein Folding; Protein Refolding; Proteins; Recombinant Proteins
PubMed: 22965925
DOI: 10.1002/biot.201200025 -
Recent Patents on Biotechnology Apr 2012Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding... (Review)
Review
Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding refers to folding back the proteins after solubilizing/unfolding the misfolded proteins of the inclusion bodies. Protein aggregation, a concentration dependent phenomenon, competes with refolding pathway. The refolding strategies largely aim at reducing aggregation and/or promoting correct folding. This review focuses on non-chromatographic strategies for refolding like dilution, precipitation, three phase partitioning and macro-(affinity ligand) facilitated three phase partitioning. The nanomaterials which disperse well in aqueous buffers are also discussed in the context of facilitating protein refolding. Apart from general results with these methods, the review also covers the use of non-chromatographic methods in protein refolding in the patented literature beyond 2000. The patented literature generally describes use of cocktail of additives which results in increase in refolding yield. Such additives include low concentration of chaotropic agents, redox systems, ions like SO4(2-) and Cl-, amines, carboxylic acids and surfactants. Some novel approaches like use of a "pressure window" or ionic liquids for refolding and immobilized diselenide compounds for ensuring correct -S-S- bonds pairing have also been discussed in various patents. In most of the patented literature, focus naturally has been on refolding in case of pharmaceutical proteins.
Topics: Chemistry Techniques, Analytical; Inclusion Bodies; Patents as Topic; Protein Refolding; Proteins; Recombinant Proteins
PubMed: 22420882
DOI: 10.2174/187220812799789172 -
Journal of Chromatography. A Apr 2023Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size...
Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size exclusion chromatography (SEC) has been extensively used for MAR because of its ability to load and refold proteins at high concentrations. However, the SEC-based batch MAR processes have the disadvantages of requiring longer columns for better separation and product dilution due to a high column-to-sample volume ratio. In this work, a modified operational scheme is developed for continuous MAR of L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC). The volumetric productivity of the modified SEC-PCC process is 6.8-fold higher than the batch SEC process. In addition, the specific buffer consumption decreased by 5-fold compared to the batch process. However, the specific activity of the refolded protein (110-130 IU/mg) was less due to the presence of impurities and additives in the refolding buffer. To address this challenge, a 2-stage process was developed for continuous refolding and purification of IBs using different matrices in sequential PCCs. The performance of the 2-stage process is compared with literature reports on single-stage IMAC-PCC and conventional pulse dilution processes for refolding L-asparaginase IBs. The 2-stage process resulted in a refolded protein with enhanced specific activity (175-190 IU/mg) and a high recovery of 84%. The specific buffer consumption (6.2 mL/mg) was lower than the pulse dilution process and comparable to the single-stage IMAC-PCC. A seamless integration of the two stages would considerably increase the throughput without compromising other parameters. High recovery, throughput, and increased operational flexibility make the 2-stage process an attractive option for protein refolding.
Topics: Asparaginase; Chromatography, Gel; Countercurrent Distribution; Protein Folding; Protein Refolding; Proteins; Recombinant Proteins; Inclusion Bodies
PubMed: 37003075
DOI: 10.1016/j.chroma.2023.463938 -
Biotechnology Advances Dec 2022Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be... (Review)
Review
Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be further purified to obtain pure and active biologicals. This work focusses on refolding as a significant process step during biopharmaceutical manufacturing with an industrial perspective. A theoretical and historical background on protein refolding gives the reader a starting point for further insights into industrial process development. Quality requirements on IBs as starting material for refolding are discussed and further economic and ecological aspects are considered with regards to buffer systems and refolding conditions. A process development roadmap shows the development of a refolding process starting from first exploratory screening rounds to scale-up and implementation in manufacturing plant. Different aspects, with a direct influence on yield, such as the selection of chemicals including pH, ionic strength, additives, etc., and other often neglected aspects, important during scale-up, such as mixing, and gas-fluid interaction, are highlighted with the use of a quality by design (QbD) approach. The benefits of simulation sciences (process simulation and computer fluid dynamics) and process analytical technology (PAT) for seamless process development are emphasized. The work concludes with an outlook on future applications of refolding and highlights open research inquiries.
Topics: Biological Products; Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins
PubMed: 36252795
DOI: 10.1016/j.biotechadv.2022.108050 -
Current Opinion in Biotechnology Apr 2001Inclusion body refolding processes are poised to play a major role in the production of recombinant proteins. Improving renaturation yields by minimizing aggregation and... (Review)
Review
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 for simultaneous solubilization and renaturation.
Topics: Disulfides; Drug Industry; Inclusion Bodies; Oxidation-Reduction; Pressure; Protein Folding; Protein Renaturation; Recombinant Proteins
PubMed: 11287238
DOI: 10.1016/s0958-1669(00)00200-7 -
Chemical Communications (Cambridge,... Dec 2009We employed a urease-catalyzed reaction to gradually remove a high concentration of a chaotropic agent (urea) from a denatured protein solution and demonstrated that...
We employed a urease-catalyzed reaction to gradually remove a high concentration of a chaotropic agent (urea) from a denatured protein solution and demonstrated that efficient protein refolding can be achieved by the urease-catalyzed reaction, without large-volume dilution.
Topics: Enzymes, Immobilized; Methods; Protein Folding; Protein Renaturation; Solutions; Urea; Urease
PubMed: 19921028
DOI: 10.1039/b916225a -
Protein Expression and Purification Jan 2004In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important... (Review)
Review
In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important polypeptides that are overexpressed in Escherichia coli. This review focuses on protein refolding methods based on column procedures because recent advances in chromatographic refolding have shown promising results.
Topics: Chaperonins; Chromatography, Gel; Escherichia coli; Humans; Inclusion Bodies; Liposomes; Polyethylene Glycols; Protein Folding; Protein Renaturation; Urea
PubMed: 14680955
DOI: 10.1016/j.pep.2003.08.023 -
Journal of Chromatography. A Nov 2010The application of ion-exchange (IEX) chromatography to protein refolding (IExR) has been successfully proven, as supported by various studies using different model...
The application of ion-exchange (IEX) chromatography to protein refolding (IExR) has been successfully proven, as supported by various studies using different model proteins, ion-exchange media and flow configurations. Ion-exchange refolding offers a relatively high degree of process intensification, represented by the possibility of performing protein refolding, product purification and product concentration, in one unit operation. Besides its high degree of process intensification, IExR offers an additional set of key advantages including: spatial isolation of the bound protein molecules and the controllable change in chemical composition using gradients. Despite of the acknowledgement of the former advantages, the lack of mechanistic understanding on how they influence the process performance of the ion-exchange refolding reactor, limits the ability to exploit them in order to optimize the performance of the unit. This paper presents a quantitative analysis that assesses the effect that the spatial isolation and the urea gradient, have on the IExR performance, judged on the basis of the refolding yield (Y(N)) and the fractional mass recovery (f(Prot,Rec)). Additionally, this work discusses the effect of the protein load, the protein loading state (i.e., native, denatured, denatured and reduced (D&R)) and the adsorbent type on f(Prot,Rec). The presented work shows: (1) that the protein load has a direct effect on f(Prot,Rec), and the magnitude of this effect depends on the loading state of the protein solution and the adsorbent type; (2) that irrespectively of the type of adsorbent used, the saturation capacity of a denatured protein is less than the native protein and that this difference can be linked to differences in accessible binding surface area; (3) that there is a clear correlation between fractional surface coverage (θ) and f(Prot,Rec), indicating that the former could serve as a good descriptor to assess spatial isolation, and (4) that the urea gradient has a direct link with the variations on the refolding yield, and this link can be quantitatively estimated using as descriptor the urea gradient slope (ξ). Overall, the information provided in this paper aims at the eventual development of rational design or selection strategies of ion-exchange media for the satisfactory and successful refolding of a target protein.
Topics: Adsorption; Chromatography, Ion Exchange; Models, Chemical; Muramidase; Protein Refolding; Proteins; Sepharose; Surface Properties; Urea
PubMed: 20933240
DOI: 10.1016/j.chroma.2010.09.044