Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion

Proteolytic resistance, as conferred by protein aggregation into inclusion bodies, has not been explored in detail. We have investigated the eventual digestion of several closely-related proteins, namely six insertional and two fusion mutants of the homotrimeric bacteriophage P22 tailspike (TSP) protein. When over-produced in E. coli, all these polypeptides form inclusion bodies accompanied by only traces of soluble protein. The mutations introduced in TSP impaired its degradation and enhanced its half live up to ten-fold, without affecting protein solubility. This indicates that protein properties other than solubility, are the main determinants of susceptibility to proteolysis. In addition, the analysis of the degradation fragments strongly suggests that the aggregated TSP polypeptides undergo a site-limited proteolytic attack, and that their complete digestion occurs through an in situ cascade cleavage process.     

Confronting high-throughput protein refolding using high pressure and solution screens

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.     

Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms.

Inclusion bodies formed by two closely related hybrid proteins, namely VP1LAC and LACVP1, have been compared during their building in Escherichia coli. Features of these proteins are determinant of aggregation rates and protein composition of the bodies, generating insoluble particles with distinguishable volume evolution. Interestingly, in LACVP1 and less perceptibly in VP1LAC bodies, an important fraction of the aggregated polypeptide is lost at a given stage of body construction. Stable degradation intermediates of the more fragile LACVP1 are concomitantly found embedded in the bodies. When recombinant protein synthesis is arrested in growing cells, the amount of aggregated protein drops while the amount of soluble protein undergoes a sudden rise before proteolysis. This indicates an architectural plasticity during the in vivo building of the studied inclusion bodies by a dynamic transition between soluble and insoluble forms of the recombinant proteins involved. During this transition, protease-sensitive polypeptides can suffer an efficient proteolytic attack and the resulting fragments further aggregate as inclusion body components.     

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.     

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.     

Monoclonal antibody epitope mapping describes tailspike beta-helix folding and aggregation intermediates

There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine alpha-tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel beta-helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the beta-helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.     

In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process

Misfolded proteins undergo a preferent degradation ruled by the housekeeping bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases. The susceptibility of aggregated polypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells. We have studied here the in vitro proteolysis of beta-galactosidase fusion proteins by trypsin treatment of purified inclusion bodies. A cascade digestion process similar to that occurring in vivo has been observed in the insoluble fraction of the digestion reaction. This suggests that major protease target sites are not either lost or newly generated by protein precipitation and that the digestion occurs in situ probably on solvent-exposed surfaces of inclusion bodies. In addition, the sequence of the proteolytic attack is influenced by protein determinants other than amino acid sequence, the early digestion steps having a dramatic influence on the further cleavage susceptibility of the intermediate degradation fragments. These observations indicate unexpected conformational changes of inclusion body proteins during their site-limited digestion, that could promote protein release from aggregates, thus partially accounting for the plasticity of in vivo protein precipitation and solubilization in bacteria.     

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.     

Routes to active proteins from transformed microorganisms.

Over-expression of recombinant proteins in microbial hosts results in the formation of active soluble protein or of insoluble aggregates (inclusion bodies). Efficient in vitro refolding strategies have been developed to reactivate inactive proteins from inclusion bodies. Co-expression of molecular chaperones may provide a tool to promote correct structure formation of recombinant proteins in vivo.     

Localization of functional polypeptides in bacterial inclusion bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a beta-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular beta-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases--the apical domain of GroEL (mini-chaperone) and two oxidoreductases (DsbA and DsbC) from Escherichia coli--were studied in refolding a protein with immunoglobulin fold (immunoglobulin-folded protein) that had been produced as inclusion bodies in E.coli. The foldases promoted the refolding of single-chain antibody fragments from denaturant-solubilized and reduced inclusion bodies in vitro, and also effectively functioned as alternatives for labilizing agent and oxidizing reagent in the stepwise dialysis system. Immobilization of the oxidoreductases enhanced refolding and recovery of functional single-chain antibody in the dialysis system, suggesting that immobilized oxidoreductases can be used as an effective additive for refolding immunoglobulin-folded proteins in vitro.     

Review: Protein refolding and inactivation during bioseparation: Bioprocessing implications

The recombinant production of proteins leads to inclusion bodies which contain aggregated proteins in active, partially active, and inactive conformational states. These aggregated proteins must be extracted from the inclusion bodies, unfolded, and carefully refolded to the active and the stable conformational state. Mechanistic models for protein refolding are briefly presented. Different strategies and protocols are presented that lead to the active and stable protein conformational state. The techniques presented include chaperonin-assisted refolding, amino acid substitution, polyethylene glycolassisted refolding, protein refolding in reverse micelles, and antibody-assisted refolding of proteins. The techniques presented together provide a reasonable framework of the state-of-the-art and may be carefully applied to the bioseparation of other proteins and biological macromolecules of interest. (c) 1995 John Wiley & Sons, Inc.     

蛋白质的排阻色谱复性的新进展

外源蛋白在大肠杆菌中高效表达时 ,常常形成不溶的、无活性的包涵体 ,包涵体蛋白的复性是重组蛋白生产过程中的一个技术难题。排阻色谱 (sizeexclusionchromatography ,SEC)用于蛋白复性是一种较新的、适用于任何一种蛋白的方法 ,与常用的稀释复性法相比 ,它能在高的起始蛋白浓度下对蛋白进行复性 ,活性回收率较高 ,同时又能使目标蛋白得到一定程度的纯化。对使用SEC复性的进展进行了评述 ,其内容包括SEC复性的原理及其复性过程中的影响因素 ,并对其未来发展进行了展望。     

Localization of Functional Polypeptides in Bacterial Inclusion Bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a β-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular β-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

Role of arginine in protein refolding, solubilization, and purification

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.     

Ultra scale‐down of protein refold screening in microwells: Challenges,solutions and application

Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF‐1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale‐up using hen egg‐white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell‐based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body‐derived protein products. Biotechnol. Bioeng. 2009;103: 329–340. © 2008 Wiley Periodicals, Inc.     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The beta-strand-rich single chain Fv fragment (scFv) and alpha-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is alpha-helix rich, are predominated by alpha-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the alpha-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.