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Proceedings of the National Academy of... Nov 2022The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and...
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 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 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.
Topics: Cytosol; Escherichia coli; Escherichia coli Proteins; Heat-Shock Proteins; Molecular Chaperones; Protein Refolding; Proteome
PubMed: 36417429
DOI: 10.1073/pnas.2210536119 -
Protein Expression and Purification Mar 2022Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several...
Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several parasites that cause diseases of great interest to public health, mostly without adequate treatment and included in the neglected tropical diseases category. One of them is Trypanosoma cruzi which causes Chagas disease and has two metacaspases involved in its PCD: TcMCA3 and TcMCA5. Their roles seemed different in PCD, TcMCA5 appears as a proapoptotic protein negatively regulated by its C-terminal sequence, while TcMCA3 is described as a cell cycle regulator. Despite this, the precise role of TcMCA3 and TcMCA5 and their atomic structures remain elusive. Therefore, developing methodologies to allow investigations of those metacaspases is relevant. Herein, we produced full-length and truncated versions of TcMCA5 and applied different strategies for their folded recombinant production from E. coli inclusion bodies. Biophysical assays probed the efficacy of the production method in providing a high yield of folded recombinant TcMCA5. Moreover, we modeled the TcMCA5 protein structure using experimental restraints obtained by XLMS. The experimental design for novel methods and the final protocol provided here can guide studies with other metacaspases. The production of TcMCA5 allows further investigations as protein crystallography, HTS drug discovery to create potential therapeutic in the treatment of Chagas' disease and in the way to clarify how the PCD works in the parasite.
Topics: Caspases; Protein Domains; Protein Refolding; Protozoan Proteins; Recombinant Proteins; Trypanosoma cruzi
PubMed: 34728367
DOI: 10.1016/j.pep.2021.106007 -
Microbial Cell Factories Jan 2019The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant...
BACKGROUND
The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein.
RESULTS
It was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation-refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer.
CONCLUSIONS
Maltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.
Topics: Antigen-Antibody Reactions; Circular Dichroism; Escherichia coli; Inclusion Bodies; Maltose-Binding Proteins; Protein Denaturation; Protein Refolding; Recombinant Proteins; Single-Chain Antibodies; Solubility
PubMed: 30642336
DOI: 10.1186/s12934-019-1053-9 -
Cell Stress & Chaperones Jan 2019Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many...
Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.
Topics: Animals; Disease; Evolution, Molecular; HSP70 Heat-Shock Proteins; Humans; Protein Aggregates; Protein Domains; Protein Refolding
PubMed: 30478692
DOI: 10.1007/s12192-018-0948-4 -
Archives of Biochemistry and Biophysics Aug 2015The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival... (Review)
Review
The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologs of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed.
Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Endopeptidase Clp; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation; HSP110 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Humans; Protein Aggregates; Protein Conformation; Protein Multimerization; Protein Refolding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Species Specificity
PubMed: 26159839
DOI: 10.1016/j.abb.2015.07.006 -
PloS One 2019In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion...
In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.
Topics: Dengue Virus; Hydrogen-Ion Concentration; Hydrostatic Pressure; Protein Refolding; Recombinant Proteins; Viral Nonstructural Proteins
PubMed: 30682103
DOI: 10.1371/journal.pone.0211162 -
Sub-cellular Biochemistry 2015The denaturation of protein by pressure has been generally well known since the findings of the perfect coagulation of egg white by a pressure of 7,000 atm within... (Review)
Review
The denaturation of protein by pressure has been generally well known since the findings of the perfect coagulation of egg white by a pressure of 7,000 atm within 30 min by Bridgman (J Biol Chem 19:511-512, 1914), and Kiyama and Yanagimoto (Rev Phys Chem Jpn 21:41-43, 1951) confirmed that the coagulation occurs above 3,880 kg cm(-2). Grant et al. (Science 94:616, 1941) and Suzuki and Kitamura (Abstracts of 30th annual meeting of Japanese Biochemical Society, 1957) found that SH groups are detected at the compressed sample of ovalbumin. On the other hand, Johnson and Campbell (J Cell Comp Physiol 26:43-49, 1945), Tongur (Kolloid Zhur 11:274-279, 1949; Biokhimiya 17:495-503, 1952) and Suzuki et al. (Mem Res Inst Sci Eng Ritsumeikan Univ 3:1-4, 1958) reported that the thermal denaturation of proteins is retarded in a few examples by the low pressure of about 1,000 atm. Before 1960, the studies of denaturation under high pressure were, however, rare and almost qualitative compared with those by heat, acid, urea and so on, so that there was no theory for the influence of hydrostatic pressure on the mechanism of denaturation. Here I review how I started experiments and analysis on pressure denaturation of proteins in early days of 1950s and 1960s in my laboratory and others.
Topics: Hydrostatic Pressure; Kinetics; Protein Denaturation; Protein Renaturation; Proteins
PubMed: 26174374
DOI: 10.1007/978-94-017-9918-8_1 -
Methods in Molecular Biology (Clifton,... 2023Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been...
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.
Topics: Temperature; Escherichia coli; Recombinant Proteins; Hot Temperature; Inclusion Bodies; Protein Folding; Protein Refolding
PubMed: 36656525
DOI: 10.1007/978-1-0716-2930-7_13 -
Materials Horizons Nov 2023Regulating protein folding including assisting folding, preventing misfolding and aggregation, and facilitating refolding of proteins are of significant importance for...
Regulating protein folding including assisting 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--nChap) with enhanced activity to facilitate protein refolding. This self--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--nChap's capability to inhibit unfolding and aggregation of client proteins, and finally facilitating refolding. Compared to traditional nanochaperones, the self--nChap significantly enhances the thermal stability of horseradish peroxidase (HRP) epicyclically under harsher conditions. Moreover, the self--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--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.
Topics: Humans; Protein Refolding; HSP70 Heat-Shock Proteins; Polymers
PubMed: 37843027
DOI: 10.1039/d3mh00619k -
International Journal of Biological... Apr 2020Despite polyphenols having had proven roles as amyloid alleviators their service has rarely been made use of in protein refolding/renaturation thus far, where...
Despite polyphenols having had proven roles as amyloid alleviators their service has rarely been made use of in protein refolding/renaturation thus far, where aggregation can be a major competing pathway. TGFβ3, expressed in inclusion bodies, is a classical example of a protein prone to high rate of aggregation severely limiting its refolding yield owing to its large cysteine content and structural complexity. Here, we have used various polyphenols (EGCG, baicalein, myricetin) either alone or in combination with the pseudo-chaperone beta cyclodextrin, in the refolding buffer. With the help of non-reducing SDS PAGE and size exclusion chromatography, we showed that refolding in the presence of baicalein or EGCG along with βCD indeed increase the yield of the native protein in a time dependent manner. EGCG expedites the refolding process giving a maximum increase of the refolding yield within 24 h while baicalein takes as long as 48 h for the same. The mechanism of mode of actions of polyphenols during refolding was further delineated by ITC. The effect of polyphenols on the aggregation kinetics and stability of native TGFβ3 were also explored. Thus these small molecules provide a promising alternate route in increasing the yield of aggregation prone proteins during refolding.
Topics: Kinetics; Polyphenols; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Folding; Protein Multimerization; Protein Refolding; Protein Stability; Spectrum Analysis; Transforming Growth Factor beta
PubMed: 31945435
DOI: 10.1016/j.ijbiomac.2020.01.024