A rational design for hepatitis B virus X protein refolding and bioprocess development guided by second virial coefficient studies

The hepatitis B virus X (HBx) protein is well known for its role in hepatitis B virus infection that often leads to hepatocellular carcinoma. Despite the clinical importance of HBx, there is little progress in anti-HBx drug development strategies due to shortage of HBx from native sources. Consistent expression of HBx as insoluble inclusion bodies within various expression systems has largely hindered HBx manufacturing via economical biosynthesis routes. Confronted by this roadblock, this study aims to quantitatively understand HBx protein behaviour in solution that will guide the rational development of a refolding-based bioprocess for HBx production. Second virial coefficient (SVC) measurements were employed to study the effects of varying physicochemical parameters on HBx intermolecular protein interaction. The SVC results suggest that covalent HBx aggregates play a key role in protein destabilisation during refolding. The use of an SVC-optimised refolding environment yielded bioactive and soluble HBx proteins from the denatured–reduced inclusion body state. This study provides new knowledge on HBx solubility behaviour in vitro, which is important in structure–function elucidation behaviour of this hydrophobic protein. Importantly, a rational refolding-based Escherichia coli bioprocess that can deliver purified and soluble HBx at large scale is successfully developed, which opens the way for rapid preparation of soluble HBx for further clinical and characterisation studies.     

REFOLD: an analytical database of protein refolding methods

The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. To bring clarity to the large and widespread quantity of published protein refolding data, we have recently established the REFOLD database (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Refolding methods are currently published in many different formats and resources--REFOLD provides a standardized system for the structured reporting and presentation of these data. Furthermore, data in REFOLD are readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information may in turn serve to facilitate the rational design and development of new refolding protocols for novel proteins. There are approximately 200 proteins currently listed in REFOLD, and it is anticipated that with the continued contribution of data by researchers this number will grow significantly, thus strengthening the emerging trends and patterns and making this database a valuable tool for the scientific community.     

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。     

Refolding single-chain antibody (scFv) using lauroyl-L-glutamate as a solubilization detergent and arginine as a refolding additive

Therapeutic potential of immunoconjugates has opened a new window for antibody-based biopharmaceuticals. Greater tissue penetration and hence enhanced cell toxicity are obtained with a smaller version of antibodies. While the whole antibody can be readily produced via mammalian expression system, antibody fragments often require refolding of insoluble proteins. Here we report a new refolding method for antibody fragments using a novel amino acid-based detergent as a solubilizing agent and arginine-assisted refolding. Inclusion bodies of antibody fragments were solubilized by 2.5% lauroyl-L-Glu (C12-L-Glu) and successfully refolded by multi-step dilution into a buffer solution containing arginine hydrochloride and thiol/disulfide-exchange reagents. Adjustment of temperature was found to be critical for increase in the refolding yield. Although each protein requires appropriate optimization, solubilization by C12-L-Glu and dilution refolding assisted by arginine can generate the native, functional antibody fragments. The procedure should enable us to utilize bacterial expression systems for the large-scale manufacturing.     

Current status of technical protein refolding

The expression of heterologous proteins in microbial hosts frequently leads to the formation of insoluble aggregates. To fully exploit the production capacity of the cells, efficient strategies for further processing have to be developed. While in lab scale matrix assisted refolding techniques, especially of histidine-tagged proteins have become very popular, in production scale refolding by dilution is still predominant due to its simplicity. However scaling up dilution processes leads to large volumes and low protein concentration. This is a heavy burden both for liquid handling and for subsequent downstream processing steps. Process development aims to operate at uniform, reproducible conditions, to reduce costs to a minimum and to guarantee the required quality of the product. The general refolding kinetics, exploration of appropriate refolding conditions are reviewed. The major refolding operations such as dilution, matrix assisted refolding, pressure driven refolding or continuous refolding applications are discussed in view of industrial applicability.     

High-throughput identification of refolding conditions for LXRbeta without a functional assay

The expression of human genes in bacteria is often one of the most efficient systems for generating proteins for drug discovery efforts. However, expression of mammalian cDNAs in Escherichia coli often results in the production of protein that is insoluble and misfolded and thus requires the development of a successful refolding procedure to generate active protein. To accelerate the process of developing protein refolding protocols, we have developed a semi-automated screening and assay system that utilizes an incomplete factorial approach to sample a large "space" of refolding conditions based on parameters known to influence protein stability and solubility. Testing of these conditions is performed readily in a 96-well plate format with minimal sample manipulation. The folded protein is resolved and detected using an HPLC equipped with a mini-column and a highly sensitive fluorescence detector. This simple method requires only a small amount of protein for the entire screen (<1 mg), and most importantly, a functional assay is not required to assess the refolding yields. Here, we validate the utility of this screening system using two model proteins, IL13 and MMP13, and demonstrate its successful application to the refolding of our target protein, the ligand-binding domain of rat liver X receptor beta.     

Folding aggregated proteins into functionally active forms

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.     

Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria

The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact beta-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.     

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.     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。     

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (相似文献   

A screening system for the identification of refolding conditions for a model protein kinase, p38alpha

Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38α, and applied this system to denatured p38α derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38α can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38α protein kinase.     

Effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor (Cys17Ser) in aqueous two-phase systems containing chelated metal ions

High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)-dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.     

Preparative protein refolding

The rapid provision of purified native protein underpins both structural biology and the development of new biopharmaceuticals. The dominance of Escherichia coli as a cellular biofactory depends on technology for solubilizing and refolding proteins that are expressed as insoluble inclusion bodies. Such technology must be scale invariant, easily automated, generic for a broad range of similar proteins and economical. Refolding methods relying on denaturant dilution and column-based approaches meet these criteria. Recent developments, particularly in column-based methods, promise to extend the range of proteins that can be refolded successfully. Developments in preparing denatured purified protein and in the analysis of protein refolding products promise to remove bottlenecks in the overall process. Combined, these developments promise to facilitate the rapid and automated determination of appropriate refolding conditions and to simplify scale-up.     

In vitro folding of alpha-helical membrane proteins

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.     

Matrix-assisted refolding of oligomeric small heat-shock protein Hsp26

Recombinant gene expression in the prokaryotic host Escherichia coli is of general interest for both biotechnology and basic research. Use of E. coli is inexpensive and advantageous due to the fully developed genetic accessibility. However, often insoluble target protein (inclusion body) accumulates in the cell. Especially when producing eukaryotic or disulfide bridged proteins in E. coli, inclusion body formation is observed. Nonetheless, insoluble protein can be regained and refolded in vitro. Commonly, renaturation of proteins is accomplished by methods involving dilution and/or dialysis. An interesting alternative is matrix-assisted refolding in which the denatured protein is refolded in the immobilized state. Here, matrix-assisted refolding was applied to refold a double cysteine variant of Hsp26, a small heat-shock protein from Saccharomyces cerevisiae which was insoluble after biosynthesis in E. coli BL21 (DE3) cells. This oligomeric protein was efficiently recovered from the insoluble fraction and refolded to its native oligomeric and chaperone-active state using ion exchange and size exclusion chromatography.     

In vitro folding of alpha-helical membrane proteins

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.     

The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production

The biotechnological production of recombinant proteins is challenged by processes that decrease the yield, such as protease action, aggregation, or misfolding. Today, the variation of strains and vector systems or the modulation of inducible promoter activities is commonly used to optimize expression systems. Alternatively, aggregation to inclusion bodies may be a desired starting point for protein isolation and refolding. The discovery of the twin-arginine translocation (Tat) system for folded proteins now opens new perspectives because in most cases, the Tat machinery does not allow the passage of unfolded proteins. This feature of the Tat system can be exploited for biotechnological purposes, as expression systems may be developed that ensure a virtually complete folding of a recombinant protein before purification. This review focuses on the characteristics that make recombinant Tat systems attractive for biotechnology and discusses problems and possible solutions for an efficient translocation of folded proteins.     

Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK

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.