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.     

Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques

Background and Aims

In a previous report, we characterized a condition of gluten sensitivity in juvenile rhesus macaques that is similar in many respects to the human condition of gluten sensitivity, celiac disease. This animal model of gluten sensitivity may therefore be useful toward studying both the pathogenesis and the treatment of celiac disease. Here, we perform two pilot experiments to demonstrate the potential utility of this model for studying intestinal permeability toward an immunotoxic gluten peptide and pharmacological detoxification of gluten in vivo by an oral enzyme drug candidate.

Methods

Intestinal permeability was investigated in age-matched gluten-sensitive and control macaques by using mass spectrometry to detect and quantify an orally dosed, isotope labeled 33-mer gluten peptide delivered across the intestinal epithelium to the plasma. The protective effect of a therapeutically promising oral protease, EP-B2, was evaluated in a gluten-sensitive macaque by administering a daily gluten challenge with or without EP-B2 supplementation. ELISA-based antibody assays and blinded clinical evaluations of this macaque and of an age-matched control were conducted to assess responses to gluten.

Results

Labeled 33-mer peptide was detected in the plasma of a gluten-sensitive macaque, both in remission and during active disease, but not in the plasma of healthy controls. Administration of EP-B2, but not vehicle, prevented clinical relapse in response to a dietary gluten challenge. Unexpectedly, a marked increase in anti-gliadin (IgG and IgA) and anti-transglutaminase (IgG) antibodies was observed during the EP-B2 treatment phase.

Conclusions

Gluten-sensitive rhesus macaques may be an attractive resource for investigating important aspects of celiac disease, including enhanced intestinal permeability and pharmacology of oral enzyme drug candidates. Orally dosed EP-B2 exerts a protective effect against ingested gluten. Limited data suggest that enhanced permeability of short gluten peptides generated by gastrically active glutenases may trigger an elevated antibody response, but that these antibodies are not necessarily causative of clinical illness.     

Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy

Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.     

A New Artificial Chaperone for Protein Refolding: Sequential Use of Detergent and Alginate

A novel artificial chaperone system using a combination of detergents and alginate was developed to refold three enzymes with totally different structures. Upon dilution of denatured protein in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used alginate, a linear copolymer of d-mannuronic acid and l-guluronic acid, to initiate and complete the refolding process. The results indicated that the extent of refolding assistance for the proteins was different due to detergent structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic and hydrophobic interactions among the capturing and stripping agents used in this investigation. Based on this newly developed method, it is expected that the protein refolding operation can be achieved easily, cheaply and efficiently.     

A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties

Background and Aims

Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem.

Methods

We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay.

Results

ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant α2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EP-B2 under simulated gastric conditions.

Conclusions

Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases.     

Refolding of superoxide dismutase by ion-exchange chromatography

A new ion-exchange chromatography process was developed for refolding of iron superoxide dismutase (Fe-SOD) produced in Escherichia coli as an inclusion body. After adsorption on an ion-exchange matrix, the denatured protein was eluted by gradient decrease of urea concentration and pH of the elution buffer. The dual gradient allowed the denatured protein to refold to its correct native conformation with return of biological activity. Compared with the traditional dilution, refolding process, the new process increased the refolding yield five-fold. The process could also be carried out at high protein concentration to decrease the solution volume after refolding.     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

Refolding strategies for ketosteroid isomerase following insoluble expression in Escherichia coli

Dilution and column-based protein refolding techniques are compared for refolding Delta 5-3-ketosteroid isomerase (KSI) with a C-terminus his6-tag. Column refolding was performed by removing the denaturant while the protein was adsorbed in an immobilized metal affinity chromatography column. Both dilution refolding and a single-step column-based refolding strategy were optimized to maximize the recovery of KSI enzyme activity, and achieved refolding yields of 87% and 70% respectively. It was found that the column-based refolding yield was reduced at higher adsorbed protein concentrations. An elution gradient with increasing imidazole concentration was used to selectively elute the biologically active KSI protein following column refolding, with high molecular weight KSI aggregates retained in the column. An iterative column-refolding process was then developed to denature and refold protein retained in the column, which significantly increased the refolding yield at high-adsorbed protein concentrations. Repetition of the column refolding operation increased the refolding yield from 50% to 75% for protein adsorbed at a concentration of 2.9 mg/mL of adsorbent. Although for the KSI protein column-based refolding did not improve the overall refolding yield compared to dilution refolding, it may still be advantageous due to the ease of integration with purification operations, increased control over the refolding conditions, and the ability to segregate refolded protein from inactive aggregates during elution.     

Immobilized Triton X-100-assisted refolding of Green Fluorescent Protein-Tobacco Etch Virus protease fusion protein using β-cyclodextrin as the eluent

A new protein refolding technique based on the use of the non-charged detergent Triton X-100 immobilized to the cross-linked agarose gel Sepharose High Performance has been developed. The new solid phase was used in combination with soluble β-cyclodextrin (β-CD) to refold recombinant Green Fluorescent Protein fused to Tobacco Etch Virus protease (GFPTEVP) expressed as inclusion bodies in E. coli. Previous attempts to refold recombinant GFPTEVP by dilution had failed. In the new procedure a column packed with Triton X-100-coupled Sepharose High Performance was used to capture unfolded GFPTEVP followed by elution using an increasing β-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 μg/ml. In contrast, dilution refolding of GFPTEVP at 200 μg/ml refolding buffer resulted in only 4.7% of native protein.     

On-column refolding of recombinant chemokines for NMR studies and biological assays

We have applied an efficient solid-phase protein refolding method to the milligram scale production of natively folded recombinant chemokine proteins. Chemokines are intensely studied proteins because of their roles in immune system regulation, response to inflammation, fetal development, and numerous disease states including, but not limited to, HIV-1/AIDS, cancer metastasis, Crohn's disease, asthma and arthritis. Many investigators use recombinant chemokines for research purposes, however these proteins partition almost exclusively to the inclusion body fraction when produced in Escherichia coli. A major hurdle is to correctly refold the chemokine and oxidize the two highly conserved disulfide bonds found in nearly all chemokines. Conventional methods for oxidation and refolding by dialysis or extreme dilution are effective but slow and yield large volumes of dilute chemokine. Here we use an on-column approach for rapid refolding and oxidation of four chemokines, CXCL12/SDF-1alpha (stromal cell-derived factor-1alpha), CCL5/RANTES, XCL1/lymphotactin, and CX3CL1/fractalkine. NMR spectra of SDF-1alpha, RANTES, lymphotactin, and fractalkine indicate these chemokines adopt native structures. On-column refolded SDF-1alpha is fully active in an intracellular calcium flux assay. Our success with multiple SDF-1alpha mutants and members of all four chemokine subfamilies suggests that on-column refolding is a robust method for preparative-scale production of recombinant chemokine proteins.     

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 of the recombinant protein Sm29, a step toward the production of the vaccine candidate against schistosomiasis

Schistosomiasis is an important parasitic disease, with about 240 million people infected worldwide. Humans and animals can be infected, imposing an enormous social and economic burden. The only drug available for chemotherapy, praziquantel, does not control reinfections, and an efficient vaccine for prophylaxis is still missing. However, the tegumental protein Sm29 of Schistosoma mansoni was shown to be a promising antigen to compose an anti-schistosomiasis vaccine. Though, recombinant Sm29 is expressed in Escherichia coli as insoluble inclusion bodies requiring an efficient process of refolding, thus, hampering its production in large scale. We present in this work studies to refold the recombinant Sm29 using high hydrostatic pressure, a mild condition to dissociate aggregated proteins, leading to refolding on a soluble conformation. Our studies resulted in high yield of rSm29 (73%) as a stably soluble and structured protein. The refolded antigen presented protective effect against S. mansoni development in immunized mice. We concluded that the refolding process by application of high hydrostatic pressure succeeded, and the procedure can be scaled-up, allowing industrial production of Sm29.     

Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.     

Factors affecting protein refolding yields in a fed-batch and batch-refolding system

The refolding of recombinant protein from inclusion bodies expressed in Escherichia coli can present a process bottleneck. Yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. This article studies the effect of five factors upon the dilution refolding of protein in a twin impeller fed-batch system using refold buffer containing only the oxidized form of the redox reagent. Such a buffer is easier to prepare and more stable than a buffer containing both reduced and oxidized forms. The five factors chosen were: bulk impeller Reynolds number, mini-impeller Reynolds number, injection rate of denatured protein, redox ratio, and guanidine hydrochloride (GdHCl) concentration. A 2(5) factorial experiment was conducted at an industrially relevant protein concentration using lysozyme as the test system. The study identified that in the system used, the guanidine hydrochloride concentration, redox ratio, and injection rate were the most important factors in determining refolding yields. Two interactions were found to be important: redox ratio/guanidine hydrochloride concentration and guanidine hydrochloride concentration/injection rate. Conditions were also found at which high refolding yields could be achieved even with rapid injection and poor mixing efficiency. Therefore, a comparative assessment was carried out with minimal mixing in a simple batch-refolding mode of operation, which revealed different behavior to that of fed-batch. A graphical (windows of operation) analysis of the batch data suggested that optimal yields and productivity are obtained at high guanidine hydrochloride concentrations (1.2 M) and redox ratios of unity or greater.     

Matrix assisted refolding of proteins by ion exchange chromatography

Two different approaches of matrix assisted refolding have been evaluated and compared to conventional refolding by dilution. Bovine alpha-lactalbumin was used for the studies as model protein. It was adsorbed under denaturing conditions on an ion exchange matrix and refolding was completed on the column prior to elution or, depending on the buffer system, in the eluate. Agarose based chromatography matrices showed high capacities for the denatured alpha-lactalbumin. A positive effect on the yield of refolded protein by the matrix could be observed for Fractogel EMD DEAE and a negative for Toyopearl DEAE 650M, DEAE Sepharose FF and Q Sepharose FF. In the case of Fractogel EMD DEAE the ion exchange surface might act as a folding helper. This property may be caused by the grafted polymers. For Source 30Q only a marginal negative influence on the refolding kinetics was observed, thus the ion exchanger is only a mean for removal of chaotropic agents. Refolding on the column is characterized by a low yield but high productivity due to significant reduction of refolding time.