Development of self-cooperative nanochaperones with enhanced activity to facilitate protein refolding.

Regulating protein folding including assisting de novo folding, preventing misfolding and aggregation, and facilitating refolding of proteins are of significant importance for retaining protein's biological activities. Here, we report a mixed shell polymeric micelle (MSPM)-based self-cooperative nanochaperone (self-CO-nChap) with enhanced activity to facilitate protein refolding. This self-CO-nChap was fabricated by introducing Hsp40-mimetic artificial carriers into the traditional nanochaperone to cooperate with the Hsp70-mimetic confined hydrophobic microdomains. The artificial carrier facilitates transfer and immobilization of client proteins into confined hydrophobic microdomains, by which significantly improving self-CO-nChap's capability to inhibit unfolding and aggregation of client proteins, and finally facilitating refolding. Compared to traditional nanochaperones, the self-CO-nChap significantly enhances the thermal stability of horseradish peroxidase (HRP) epicyclically under harsher conditions. Moreover, the self-CO-nChap efficiently protects misfolding-prone proteins, such as immunoglobulin G (IgG) antibody from thermal denaturation, which is hardly achieved using traditional nanochaperones. In addition, a kinetic partitioning mechanism was devised to explain how self-CO-nChap facilitates refolding by regulating the cooperative effect of kinetics between the nanochaperone and client proteins. This work provides a novel strategy for the design of protein folding regulatory materials, including nanochaperones.

Self-Assembly Nanochaperone with Tunable Hydrophilic-Hydrophobic Surface for Controlled Protein Refolding.

Nanochaperones (nChaps) have significant potential to inhibit protein aggregation and assist in protein refolding. The interaction between nChaps and proteins plays an important role in nChaps performing chaperone-like functions, but the interaction mechanism remains elusive. In this work, a series of nChaps with tunable hydrophilic-hydrophobic surfaces are prepared, and the process of nChaps-assisted denatured protein refolding is systematically explored. It is found that an appropriate hydrophilic-hydrophobic balance on the nChap surface is critical for enhancing protein renaturation. This is because only the optimal interaction between nChap and protein can simultaneously guarantee the suitable capture and sufficient release of client proteins. The findings in this work will provide an effective reference for the design of nChaps and contribute to the development of the potential of nChaps in the future.

Chromatography assisted in-vitro refolding and purification of recombinant peptibody: Recombinant Romiplostim a case study.

In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 ± 1.5 % and a purity of over 79.73 ± 3.4 % were achieved at 25-fold dilution, 15 °C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.

Laboratory Scale Production of Complex Proteins Using Charge Complimentary Nanoenvironments.

Protein refolding is a crucial procedure in bacterial recombinant expression. Aggregation and misfolding are the two challenges that can affect the overall yield and specific activity of the folded proteins. We demonstrated the in vitro use of nanoscale "thermostable exoshells" (tES) to encapsulate, fold and release diverse protein substrates. With tES, the soluble yield, functional yield, and specific activity increased from 2-fold to >100-fold when compared to folding in its absence. On average, the soluble yield was determined to be 6.5 mg/100 mg of tES for a set of 12 diverse substrates evaluated. The electrostatic charge complementation between the tES interior and the protein substrate was considered as the primary determinant for functional folding. We thus describe a useful and simple method for in vitro folding that has been evaluated and implemented in our laboratory.

Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding.

Ring-forming AAA+ chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA+ chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.

Expression, purification and refolding of pro-MMP-2 from inclusion bodies of E. coli.

MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins.

Continuous protein refolding and purification by two-stage periodic counter-current chromatography.

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.

Unit Operation-Spanning Investigation of the Redox System.

Cytoplasmic expression of recombinant proteins requiring disulfide bridges in Escherichia coli usually leads to the formation of insoluble inclusion bodies (IBs). The reason for this phenomenon is found in the reducing environment of the cytoplasm, preventing the formation of disulfide bridges and therefore resulting in inactive protein aggregates. However, IBs can be refolded in vitro to obtain the protein in its active conformation. In order to correctly form the required disulfide bridges, cystines are fully reduced during solubilization and, with the help of an oxidizing agent, the native disulfide bridges are formed during the refolding step. Here, a protocol to identify suitable redox conditions for solubilization and refolding is presented. For this purpose, a multivariate approach spanning the unit operations solubilization and refolding is used.

Development of Solubilization and Refolding Buffers.

Overexpression of heterologous protein in prokaryotic host cells, such as Escherichia coli, usually leads to formation of inactive and insoluble aggregates known as inclusion bodies (IBs). Recovery of refolded and functionally bioactive proteins from IBs is a challenging task, and a unique condition (e.g., solubilizing and refolding buffers) for each individual protein should be experimentally obtained. Here, we present a simple protocol for development of solubilizing and refolding buffers for successful recovery of pure bioactive proteins from IBs.

Using High Pressure and Alkaline pH for Refolding.

The expression of recombinant proteins as insoluble inclusion bodies (IB) has the advantage to separate insoluble aggregates from soluble bacterial molecules, thus obtaining proteins with a high degree of purity. Even aggregated, the proteins in IB often present native-like secondary and tertiary structures, which can be maintained as long as solubilization is carried out in non-denaturing condition. High pressure solubilizes IB by weakening hydrophobic interactions, while alkaline pH solubilizes aggregates by electrostatic repulsion. The combination of high pressure and alkaline pH is effective for IB solubilization at a mild, non-denaturing condition, which is useful for subsequent refolding. Here, we describe the expression of recombinant proteins in Escherichia coli using a rich medium to obtain high expression levels, bacterial lysis, and washing of the IB to obtain products of high purity, and, finally, the solubilization and high yield of refolded proteins using high pressure and alkaline pH.

Intensification of Inclusion Body Processing via Temperature-Based Refolding.

Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.

Refolding of Proteins Expressed as Inclusion Bodies in E. coli.

Microbial-based biotherapeutics that are produced in Escherichia coli (E. coli) can be generated intracellularly in the form of inclusion bodies (IBs) or in soluble active form in periplasmic space or extracellularly. Overexpression of these biotherapeutics in E. coli leads to formation of insoluble aggregates called inclusion bodies. These IBs contain misfolded and inactive form of proteins which need to be refolded to obtain a functionally active form of proteins. Here, we discuss refolding of E. coli-based recombinant human granulocyte colony-stimulating factor (GCSF), expressed as IBs, and highlight some of the key features associated with the refolding kinetic reaction.

A proteome-wide map of chaperone-assisted protein refolding in a cytosol-like milieu.

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain's intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis-mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.

Refolding in the modern biopharmaceutical industry.

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.

Human growth hormone inclusion bodies present native-like secondary and tertiary structures which can be preserved by mild solubilization for refolding.

BACKGROUND: Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. RESULTS: In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield). CONCLUSIONS: We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.

In transcription antitermination by Qλ, NusA induces refolding of Qλ to form a nozzle that extends the RNA polymerase RNA-exit channel.

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.

Smart Protein Refolding System Based on UCST-Type Ureido Polymers.

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.

scFv-oligopeptide chaperoning system-assisted on-column refolding and purification of human muscle creatine kinase from inclusion bodies.

The formation of inclusion bodies in bacterial hosts poses a major challenge for the 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 describe a novel method for the renaturation and purification of inclusion bodies. This method combines a scFv-oligopeptide chaperoning system and an on-column refolding system to help refold human muscle creatine kinase (HCK) inclusion bodies. This method could significantly increase the activity recovery of denatured HCK inclusion bodies and provides an effective method for the production of bioactive proteins from inclusion bodies.

Expression in Escherichia coli, Refolding, and Purification of Plant Aspartic Proteases.

Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.

Solvent extraction of recombinant interferon alpha-2b from inclusion bodies and efficient refolding at high protein concentrations.

Interferon alpha-2b (IFNα-2b) is an essential cytokine widely used in hepatitis and cancer treatment. This paper presents a novel protocol for purification and efficient refolding of recombinant interferon alpha-2b (IFNα-2b) expressed as insoluble inclusion bodies in Escherichia coli. Purification of IFNα-2b from solubilized inclusion bodies was performed by solvent extraction using 2-butanol. Refolding conditions were optimized using the response surface method (RSM). Under optimized conditions, refolding yield of solvent-extracted IFNα-2b was 1.5 fold higher than refolding yield of unpurified IFNα-2b. High-concentration protein refolding was carried out by pulse-fed method, and refolding yield of 75% was achieved at a protein concentration of 300 µg ml-1. Under optimized conditions, 259.16 mg of purified IFNα-2b with the biological activity of 2.4 × 108 IU mg-1 was achieved per liter of bacterial culture. The developed protocol provides an efficient production process of high-quality IFNα-2b suitable for research and pharmaceutical applications.