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Current Protocols in Protein Science Nov 2014Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion... (Review)
Review
Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal-chelate affinity chromatography are described.
Topics: Animals; Escherichia coli; Guanidine; Humans; Inclusion Bodies; Protein Denaturation; Protein Refolding; Recombinant Proteins
PubMed: 25367010
DOI: 10.1002/0471140864.ps0605s78 -
European Journal of Clinical... May 2021In the last decades, cardiovascular diseases (CVD) have remained the first leading cause of mortality and morbidity in the world. Although several therapeutic approaches... (Review)
Review
BACKGROUND
In the last decades, cardiovascular diseases (CVD) have remained the first leading cause of mortality and morbidity in the world. Although several therapeutic approaches have been introduced in the past, the development of novel treatments remains an important research goal, which is hampered by the lack of understanding of key mechanisms and targets. Emerging evidences in recent years indicate the involvement of misfolded proteins aggregation and the derailment of protein quality control in the pathogenesis of cardiovascular diseases. Several potential interventions targeting protein quality control have been translated from the bench to the bedside to effectively employ the misfolded proteins as promising therapeutic targets for cardiac diseases, but with trivial results.
DESIGN
In this review, we describe the recent progresses in preclinical and clinical studies of protein misfolding and compromised protein quality control by selecting and reporting studies focusing on cardiovascular diseases including cardiomyopathies, cardiac amyloidosis, atherosclerosis, atrial fibrillation and thrombosis.
RESULTS
In preclinical models, modulators of several molecular targets (eg heat shock proteins, unfolded protein response, ubiquitin protein system, autophagy and histone deacetylases) have been tested in various conditions with promising results although lacking an adequate transition towards clinical setting.
CONCLUSIONS
At present, no therapeutic strategies have been reported to attenuate proteotoxicity in patients with CVD due to a lack of specific biomarkers for pinpointing upstream events in protein folding defects at a subclinical stage of the diseases requiring an intensive collaboration between basic scientists and clinicians.
Topics: Amyloidosis; Animals; Atherosclerosis; Atrial Fibrillation; Autophagy; Cardiomyopathies; Cardiovascular Diseases; Heat-Shock Proteins; Histone Deacetylases; Humans; Protein Aggregation, Pathological; Protein Folding; Protein Refolding; Proteostasis; Proteostasis Deficiencies; Thrombosis; Ubiquitination; Unfolded Protein Response
PubMed: 33527342
DOI: 10.1111/eci.13504 -
Methods in Molecular Biology (Clifton,... 2023Cytoplasmic expression of recombinant proteins requiring disulfide bridges in Escherichia coli usually leads to the formation of insoluble inclusion bodies (IBs). The...
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.
Topics: Disulfides; Escherichia coli; Oxidation-Reduction; Protein Folding; Protein Refolding; Recombinant Proteins; Solubility; Inclusion Bodies
PubMed: 36656523
DOI: 10.1007/978-1-0716-2930-7_11 -
Science Advances May 2018Precise protein folding is essential for the survival of all cells, and protein misfolding causes a number of diseases that lack effective therapies, yet the general...
Precise protein folding is essential for the survival of all cells, and protein misfolding causes a number of diseases that lack effective therapies, yet the general principles governing protein folding in the cell remain poorly understood. In vivo, folding can begin cotranslationally and protein quality control at the ribosome is essential for cellular proteostasis. We directly characterize and compare the refolding and cotranslational folding trajectories of the protein HaloTag. We introduce new techniques for both measuring folding kinetics and detecting the conformations of partially folded intermediates during translation in real time. We find that, although translation does not affect the rate-limiting step of HaloTag folding, a key aggregation-prone intermediate observed during in vitro refolding experiments is no longer detectable. This rerouting of the folding pathway increases HaloTag's folding efficiency and may serve as a general chaperone-independent mechanism of quality control by the ribosome.
Topics: Kinetics; Models, Molecular; Protein Conformation; Protein Folding; Protein Refolding; Proteins
PubMed: 29854950
DOI: 10.1126/sciadv.aas9098 -
International Journal of Biological... Jan 2021The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed...
The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed for various applications, such as drug delivery, molecular imaging, and liquid biopsy etc. However, the challenges remain to ensure protein stability and to retain the conformational state of the protein intact upon nano-conjugation. In this communication we have reported the status of stability and refolding ability of Au-NP conjugated zDHFR protein. The effect of nano-conjugation of zDHFR on the thermal stability and it's refolding from thermally denatured state have been extensively studied. Zebrafish Dihydrofolate reductase (zDHFR) is an essential enzyme which acts as a crucial part in synthesis of purine, thymidylate and various amino acids in cells. We have nano-conjugated zDHFR protein with Au-nanoparticles and studies were conducted for thermally denatured Au-NP conjugated zDHFR and compared with the non-conjugated protein. Refolding experiment of heat denatured Au-NP conjugated zDHFR was carried out to check the status of refolding and the result was compared with the non-conjugated protein. Our observation reveals that nano-conjugation stabilises the zDHFR protein against thermal denaturation. Furthermore, the nano-conjugation promotes refolding process of thermally unfolded DHFR such that the yield of refolding substantially increases.
Topics: Animals; Chemical Phenomena; Gene Expression; Gold; Kinetics; Metal Nanoparticles; Nanostructures; Protein Denaturation; Protein Folding; Protein Refolding; Protein Stability; Recombinant Proteins; Tetrahydrofolate Dehydrogenase; Thermodynamics; Zebrafish
PubMed: 33181215
DOI: 10.1016/j.ijbiomac.2020.11.053 -
Protein Expression and Purification Aug 2023MMP-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...
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.
Topics: Escherichia coli; Matrix Metalloproteinase 2; Recombinant Proteins; Bacterial Proteins; Inclusion Bodies; Protein Folding; Protein Refolding
PubMed: 37094772
DOI: 10.1016/j.pep.2023.106278 -
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 -
Nature Communications Oct 2016Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and...
Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.
Topics: Acetylation; Animals; Apoptosis; Caspases; Cell Survival; Green Fluorescent Proteins; HEK293 Cells; HSP70 Heat-Shock Proteins; Humans; Molecular Chaperones; Mutation; N-Terminal Acetyltransferase A; N-Terminal Acetyltransferase E; Protein Binding; Protein Domains; Protein Processing, Post-Translational; Protein Refolding; RNA, Small Interfering; Stress, Physiological; Zebrafish
PubMed: 27708256
DOI: 10.1038/ncomms12882 -
Methods in Molecular Biology (Clifton,... 2023Protein refolding is a crucial procedure in bacterial recombinant expression. Aggregation and misfolding are the two challenges that can affect the overall yield and...
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.
Topics: Laboratories; Protein Refolding; Static Electricity
PubMed: 37308658
DOI: 10.1007/978-1-0716-3222-2_23 -
BMC Structural Biology Apr 2017More than 7000 papers related to "protein refolding" have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these...
BACKGROUND
More than 7000 papers related to "protein refolding" have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource - "REFOLDdb" that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.
RESULTS
We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/ .
CONCLUSION
REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.
Topics: Algorithms; Databases, Protein; Humans; Internet; Protein Refolding; Proteins; User-Computer Interface
PubMed: 28438161
DOI: 10.1186/s12900-017-0074-z