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PloS One 2018RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune...
RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.
Topics: Amino Acid Sequence; Caspase Activation and Recruitment Domain; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Molecular; Nod1 Signaling Adaptor Protein; Nod2 Signaling Adaptor Protein; Protein Binding; Protein Multimerization; Protein Refolding; Receptor-Interacting Protein Serine-Threonine Kinase 2; Sequence Homology, Amino Acid; Solutions; Static Electricity
PubMed: 30352081
DOI: 10.1371/journal.pone.0206244 -
Protein Expression and Purification Dec 2020Hydrophobins are low molecular weight proteins secreted by fungi that are extremely surface-active and able to self-assemble into larger structures. Due to their unusual...
Hydrophobins are low molecular weight proteins secreted by fungi that are extremely surface-active and able to self-assemble into larger structures. Due to their unusual biochemical properties, hydrophobins are an attractive target for commercial applications such as drug emulsification and surface modification. When produced in E. coli, hydrophobins are often not soluble and need to be refolded. In this work we use SHuffle T7 Express E. coli coupled with glutathione redox buffers to produce and refold four distinct class IB hydrophobins that originate from Phanerochaete carnosa (PC1), Wallemia ichthyophaga (WI1), Serpula lacrymans (SL1), and Schizophyllum commune (SC16). Proper refolding and function of these purified hydrophobins was confirmed using nuclear magnetic resonance spectroscopy and thioflavin T assays. These results indicate that class IB hydrophobins can be consistently produced and purified from E. coli, aiding future structural and biochemical studies that require highly pure hydrophobins.
Topics: Basidiomycota; Escherichia coli; Fungal Proteins; Gene Expression; Protein Refolding; Recombinant Proteins
PubMed: 32866612
DOI: 10.1016/j.pep.2020.105732 -
Journal of Chromatography. A Jan 2022Chromatography-based refolding is emerging as a promising alternative to dilution-refolding of solubilized inclusion bodies (IBs). The advantages of this matrix-assisted...
Chromatography-based refolding is emerging as a promising alternative to dilution-refolding of solubilized inclusion bodies (IBs). The advantages of this matrix-assisted refolding (MAR) lie in its ability to reduce aggregate formation, leading to better recovery of active protein, and enabling refolding at higher protein concentration. However, batch chromatography has the disadvantage of ineffective solvent utilization, under-utilization of resin, and low throughput. In this work, we overcome these challenges by using a 3-column Periodic Counter-current Chromatographic (PCC) system for continuous refolding of IBs, formed during the production of L-asparaginase by recombinant E. coli cultures. Initial experiments were conducted in batch processes using single-column immobilized metal-affinity chromatography. Different gradient operations were designed to improve the protein loading for the single-column, batch-MAR processes. Optimized conditions, based on the batch-MAR experiments, were used for designing the continuous-MAR processes using the PCC system. The continuous-MAR experiments were carried out over 3 cycles (∼ 30 h) in the PCC system. A detailed quantitative comparison based on recovery, throughput, buffer consumption, and resin utilization was made for the three modes of operation: pulse-dilution, single-column batch-MAR, and 3-Column PCC-based continuous-MAR processes. While recovery (73%) and throughput (11 mg/h) were the highest in PCC, specific buffer consumption (6.9 ml/mg) was the least. Also, during PCC operation, resin utilization improved by 92% in comparison to the single-column batch-MAR process. These quantitative comparisons clearly establish the advantages of the continuous-MAR process over the batch-MAR and other conventional refolding techniques.
Topics: Asparaginase; Chromatography, Affinity; Countercurrent Distribution; Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins
PubMed: 34936904
DOI: 10.1016/j.chroma.2021.462746 -
Topics in Current Chemistry (Cham) Apr 2017In the last two decades, while searching for interesting applications of ionic liquids as potent solvents, their solvation properties and their general impact on... (Review)
Review
In the last two decades, while searching for interesting applications of ionic liquids as potent solvents, their solvation properties and their general impact on biomolecules, and in particular on proteins, gained interest. It turned out that ionic liquids are excellent solvents for protein refolding and crystallization. Biomolecules showed increased solubilities and stabilities, both operational and thermal, in ionic liquids, which also seem to prevent self-aggregation during solubilization. Biomolecules can be immobilized, e.g. in highly viscous ionic liquids, for particular biochemical processes and can be designed to some extent by the proper choice of the ionic liquid cations and anions, which can be characterized by the Hofmeister series.
Topics: Ionic Liquids; Models, Molecular; Molecular Structure; Protein Refolding; Proteins; Solvents
PubMed: 28176271
DOI: 10.1007/s41061-017-0110-2 -
Macromolecular Bioscience Nov 2023Nanochaperones (nChaps) have significant potential to inhibit protein aggregation and assist in protein refolding. The interaction between nChaps and proteins plays an...
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.
Topics: Humans; Protein Refolding; Molecular Chaperones; Protein Folding; Protein Denaturation
PubMed: 37463112
DOI: 10.1002/mabi.202300205 -
Biochimica Et Biophysica Acta.... Apr 2017Biosurfactants (BS) are surface-active molecules produced by microorganisms. For several decades they have attracted interest as promising alternatives to current... (Review)
Review
Biosurfactants (BS) are surface-active molecules produced by microorganisms. For several decades they have attracted interest as promising alternatives to current petroleum-based surfactants. Aside from their green profile, they have remarkably low critical micelle concentrations, reduce the air/water surface tension to very low levels and are excellent emulsifiers, all of which make them comparable or superior to their synthetic counterparts. These remarkable physical properties derive from their more complex chemical structures in which hydrophilic and hydrophobic regions are not as clearly separated as chemical surfactants but have a more mosaic distribution of polarity as well as branched or circular structures. This allows the lipopeptide surfactin to adopt spherical structures to facilitate dense packing at interfaces. They are also more complex. Glycolipid BS, e.g. rhamnolipids (RL) and sophorolipids, are produced biologically as mixtures which vary in the size and saturation of the hydrophobic region as well as modifications in the hydrophilic headgroup, such as the number of sugar groups and different levels of acetylation, leading to variable surface-active properties. Their amphiphilicity allows RL to insert easily into membranes at sub-cmc concentrations to modulate membrane structure and extract lipopolysaccharides, leading to extensive biofilm remodeling in vivo, sometimes in collaboration with hydrophobic RL precursors. Thanks to their mosaicity, even anionic BS like RL only bind weakly to proteins and show much lower denaturing potency, even supporting membrane protein refolding. Nevertheless, they can promote protein degradation by proteases e.g. by neutralizing positive charges, which together with their biofilm-combating properties makes them very promising detergent surfactants. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.
Topics: Acetylation; Cell Membrane; Emulsifying Agents; Glycolipids; Hydrophobic and Hydrophilic Interactions; Lipopeptides; Membrane Proteins; Micelles; Models, Molecular; Peptides, Cyclic; Protein Refolding; Saponins; Surface Properties; Surface Tension; Surface-Active Agents
PubMed: 27693345
DOI: 10.1016/j.bbamem.2016.09.024 -
Protein Expression and Purification Sep 2022Interferon alpha-2b (IFNα-2b) is an essential cytokine widely used in hepatitis and cancer treatment. This paper presents a novel protocol for purification and...
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. Under optimized conditions, 259.16 mg of purified IFNα-2b with the biological activity of 2.4 × 10 IU mg 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.
Topics: Escherichia coli; Inclusion Bodies; Interferon alpha-2; Protein Refolding; Recombinant Proteins; Solvents
PubMed: 35577182
DOI: 10.1016/j.pep.2022.106110 -
Scientific Reports Aug 2017Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion...
Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.
Topics: Buffers; Chromatography, Gel; Hydrogen-Ion Concentration; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Interaction Domains and Motifs; Protein Refolding; Proteins; Recombinant Proteins; Solubility
PubMed: 28839267
DOI: 10.1038/s41598-017-09687-z -
International Journal of Biological... Sep 2023In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli....
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.
Topics: Escherichia coli; Cysteine; Recombinant Proteins; Protein Refolding; Chromatography, Affinity; Disulfides; Protein Folding
PubMed: 37516226
DOI: 10.1016/j.ijbiomac.2023.126037 -
Journal of Chromatography. A Feb 2015Chromatography is the key technology in protein purification as well as in protein refolding. Taking the scientific development and technological innovation of protein... (Review)
Review
Chromatography is the key technology in protein purification as well as in protein refolding. Taking the scientific development and technological innovation of protein chromatography as the objective, this article is devoted to an overview of protein behavior at chromatographic surfaces, including protein orientation, conformational transitions (unfolding and refolding), and protein transport. Recent advances achieved by using molecular simulations as well as theoretical and experimental investigations are elaborated and discussed with emphasis on their implications to the rational design of novel chromatographic surfaces or materials and mobile phase conditions for the development of high-performance protein chromatography.
Topics: Chromatography; Protein Conformation; Protein Refolding; Protein Transport; Proteins
PubMed: 25601319
DOI: 10.1016/j.chroma.2014.12.087