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1.
In vitro folding of alpha-helical membrane proteins 总被引:8,自引:0,他引:8
Kiefer H 《Biochimica et biophysica acta》2003,1610(1):57-62
For large-scale production, as required in structural biology, membrane proteins can be expressed in an insoluble form as inclusion bodies and be refolded in vitro. This requires refolding conditions where the native form is thermodynamically stable and where nonproductive pathways leading to aggregation are avoided. Examples of successful refolding are reviewed and general guidelines to establish refolding protocols of membrane proteins are presented. 相似文献
2.
Roussel G Matagne A De Bolle X Perpète EA Michaux C 《Protein expression and purification》2012,83(2):198-204
Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination. 相似文献
3.
Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals
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Domenico Azarnia Tehran Marco Pirazzini Oneda Leka Andrea Mattarei Florigio Lista Thomas Binz Ornella Rossetto Cesare Montecucco 《Cellular microbiology》2017,19(2)
Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase–Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX‐12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase–Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone‐redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol. 相似文献
4.
Raghava S Barua B Singh PK Das M Madan L Bhattacharyya S Bajaj K Gopal B Varadarajan R Gupta MN 《Protein science : a publication of the Protein Society》2008,17(11):1987-1997
Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies. 相似文献
5.
Swietnicki W 《Current opinion in biotechnology》2006,17(4):367-372
The successful expression and purification of proteins in an active form is essential for structural and biochemical studies. With rapid advances in genome sequencing and high-throughput structural biology, an increasing number of proteins are being identified as potential drug targets but are difficult to obtain in a form suitable for structural or biochemical studies. Although prokaryotic recombinant expression systems are often used, proteins obtained in this way are typically found to be insoluble. Several experimental approaches have therefore been developed to refold these aggregated proteins into a biologically active form, often suitable for structural studies. The major refolding strategies adopt one of two approaches - chromatographic methods or refolding in free solution - and both routes have been successfully used to refold a range of proteins. Future advances are likely to involve the development of automated approaches for protein refolding and purification. 相似文献
6.
Membrane protein folding and oligomerization: the two-stage model. 总被引:27,自引:0,他引:27
We discuss the view that the folding of many, perhaps most, integral membrane proteins can be considered as a two-stage process. In stage I, hydrophobic alpha-helices are established across the lipid bilayer. In stage II, they interact to form functional transmembrane structures. This model is suggested by the nature of transmembrane segments in known structures, refolding experiments, the assembly of integral membrane protein from fragments, and the existence of very small integral membrane protein subunits. It may extend to proteins with a variety of functions, including the formation of transmembrane aqueous channels. The model is discussed in the context of the forces involved in membrane protein folding and the interpretation of sequence data. 相似文献
7.
Bryantsev AL Kurchashova SY Golyshev SA Polyakov VY Wunderink HF Kanon B Budagova KR Kabakov AE Kampinga HH 《The Biochemical journal》2007,407(3):407-417
In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails. 相似文献
8.
While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason. 相似文献
9.
Natalie Di Bartolo Emma L. R. Compton Tony Warne Patricia C. Edwards Christopher G. Tate Gebhard F. X. Schertler Paula J. Booth 《PloS one》2016,11(3)
The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes. 相似文献
10.
Continuous refolding of lysozyme with fed-batch addition of denatured protein solution 总被引:3,自引:0,他引:3
A continuous refolding method with addition of denatured protein solution in a fed-batch manner through a ceramic membrane tube was developed. Denatured and fully reduced lysozyme was continuously refolded with high refolding efficiencies. In this method, a denatured lysozyme solution was gradually added from the outer surface of the membrane tube into a refolding buffer flowing continuously inside the tube under controlled mixing conditions. The refolding efficiencies of lysozyme in this continuous refolding were higher than those in a batch dilution method. This method has applicability to large-scale downstream processes and can attain a high efficiency and protein concentration in refolding. Refolded proteins can be supplied continuously following purification steps. 相似文献
11.
Dr. Satoshi Yamaguchi Etsushi Yamamoto Teruhisa Mannen Teruyuki Nagamune Prof. Teruyuki Nagamune 《Biotechnology journal》2013,8(1):17-31
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-b 相似文献
12.
Vincent Dechavanne Nicolas Barrillat Frederic Borlat Aurélie Hermant Laurent Magnenat Mikael Paquet Bruno Antonsson Laurent Chevalet 《Protein expression and purification》2011,75(2):192-203
Production of correctly folded and biologically active proteins in Escherichia coli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS–PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein. 相似文献
13.
Edwards KL Kueltzo LA Fisher MT Middaugh CR 《Archives of biochemistry and biophysics》2001,393(1):14-21
Fibroblast growth factor one (FGF-1) exists in a molten globule (MG)-like state under physiological conditions (neutral pH, 37 degrees C). It has been proposed that this form of the protein may be involved in its atypical membrane transport properties. Macromolecular chaperones have been shown to bind to MG states of proteins as well as to be involved in protein membrane transport. We have therefore examined the effect of such proteins on the aggregation and refolding of FGF-1 to evaluate whether they might play a role in FGF-1 transport. The proposed chaperone alpha-crystallin was found to strongly inhibit the aggregation of the MG state of FGF-1. Curiously, two other proteins of similar size and charge (thyroglobulin and a monoclonal IgM immunoglobulin) with no previously reported chaperone properties were also found to have a related effect. In contrast, the chaperone GroEL/ES induced further aggregation of MG-like FGF-1 but had no effect on the native conformation. Both chaperones stimulated refolding to the native state (25 degrees C) but had no detectable effect when FGF-1 was refolded to the MG state (37 degrees C). This suggests that disordered intermediates are present in the folding pathways of the native and MG-like FGF conformations which differ from the MG-like state induced under physiological conditions. FGF-1 does, therefore, interact with molecular chaperones, although this may involve both the MG and the native states of the protein. 相似文献
14.
Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion bodies. Many distinct methods for protein refolding are now in use. However, regardless of method used, developing a reliable protein refolding protocol still requires significant optimization through trial and error. Many proteins fall into the category of "Challenging" or "Difficult to Express" and are problematic to refold using traditional chaotrope-based refolding techniques. This review discusses new methods for improving protein refolding, such as implementing high hydrostatic pressure, using small molecule additives to enhance traditional protein refolding strategies, as well as developing practical methods for performing refolding studies to maximize their reliability and utility. The strategies examined here focus on high-throughput, automated refolding screens, which can be applied to structural genomic projects. 相似文献
15.
The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more
pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function
identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to
address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression
of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate
insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons.
Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This
review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development
of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding
process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer
systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities
are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess
designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed. 相似文献
16.
Transient association of the first intermediate during the refolding of bovine carbonic anhydrase B.
Many proteins which aggregate during refolding may form transiently populated aggregated states which do not reduce the final recovery of active species. However, the transient association of a folding intermediate will result in reduced refolding rates if the dissociation process occurs slowly. Previous studies on the refolding and aggregation of bovine carbonic anhydrase B (CAB) have shown that the molten globule first intermediate on the CAB folding pathway will form dimers and trimers prior to the formation of large aggregates (Cleland, J. L.; Wang, D. I. C. Biochemistry 1990, 29, 11072-11078; Cleland, J. L.; Wang, D. I. C. In Protein Refolding; Georgiou, G., De-Bernardez-Clark, E., Eds.; ACS Symposium Series 470; American Chemical Society: Washington, DC, 1991; pp 169-179). Refolding of CAB from 5 M guanidine hydrochloride (GuHCl) was achieved at conditions ([CAB]f = 10-33 microM, [GuHCl]f = 1.0 M) which allowed complete recovery of active protein as well as the formation of a transiently populated dimer of the molten globule intermediate on the refolding pathway. A kinetic analysis of CAB refolding provided insight into the mechanism of the association phenomenon. Using the kinetic results, a model of the refolding with transient association was constructed. By adjusting a single variable, the dimer dissociation rate constant, the model prediction fit both the experimentally determined active protein and dimer concentrations. The model developed in this analysis should also be applicable to the refolding of proteins which have been observed to form aggregates during refolding. In particular, the transient association of hydrophobic folding intermediates may also occur during the refolding of other proteins.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Guillaume Roussel Eric A. Perpète André Matagne Emmanuel Tinti Catherine Michaux 《Biotechnology and bioengineering》2013,110(2):417-423
It has recently been reported that 2‐methyl‐2,4‐pentanediol (MPD) can modulate the protein‐binding properties of sodium dodecyl sulfate (SDS), turning it into a non‐denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD‐based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far‐UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20–40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417–423. © 2012 Wiley Periodicals, Inc. 相似文献
18.
Prodöhl A Dreher C Hielscher R Hellwig P Schneider D 《Protein expression and purification》2007,56(2):279-285
Folding and assembly studies with alpha-helical membrane proteins are often hampered by the absence of high-level expression systems as well as by missing suitable in vitro refolding procedures. Experimental constraints and requirements for heterologous expression and in vitro assembly of cytochrome b6 have been examined and conditions for in vitro reconstitutions of the protein have been optimized. Cytochrome b6 can serve as an excellent model system for in vitro studies on the dynamic interplay of an apo-protein and heme cofactors during assembly of a transmembrane b-type cytochrome. In vitro assembled cytochrome b6 binds two hemes with different midpoint potentials and both ferri as well as ferro heme bind to the apo-cytochrome. However, the ferro cytochrome appears to be less stable than the ferri form. 相似文献
19.
Mogk A Schlieker C Friedrich KL Schönfeld HJ Vierling E Bukau B 《The Journal of biological chemistry》2003,278(33):31033-31042
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK. 相似文献
20.
Stephane Marchal Anna Marabotti Maria Staiano Antonio Varriale Thomas Domaschke Reinhard Lange Sabato D’Auria 《PloS one》2012,7(11)
The lipocalin family is typically composed of small proteins characterized by a range of different molecular recognition properties. Odorant binding proteins (OBPs) are a class of proteins of this family devoted to the transport of small hydrophobic molecules in the nasal mucosa of vertebrates. Among OBPs, bovine OBP (bOBP) is of great interest for its peculiar structural organization, characterized by a domain swapping of its two monomeric subunits. The effect of pressure on unfolding and refolding of native dimeric bOBP and of an engineered monomeric form has been investigated by theoretical and experimental studies under pressure. A coherent model explains the pressure-induced protein structural changes: i) the substrate-bound protein stays in its native configuration up to 330 MPa, where it loses its substrate; ii) the substrate-free protein dissociates into monomers at 200 MPa; and iii) the monomeric substrate-free form unfolds at 120 MPa. Molecular dynamics simulations showed that the pressure-induced tertiary structural changes that accompany the quaternary structural changes are mainly localized at the interface between the monomers. Interestingly, pressure-induced unfolding is reversible, but dimerization and substrate binding can no longer occur. The volume of the unfolding kinetic transition state of the monomer has been found to be similar to that of the folded state. This suggests that its refolding requires relatively large structural and/or hydrational changes, explaining thus the relatively low stability of the monomeric form of this class of proteins. 相似文献