Dilution Refolding Research Articles

Heterologous expression, refolding, and characterization of a calcium-independent phospholipase A1 from Streptomyces albidoflavus

Phospholipase A1 (PLA1) is a member of the hydrolase family with applications in various fields, especially in the food industry. A calcium-independent PLA1 from Streptomyces albidoflavus was expressed in E. coli BL21 (DE3) in this study. The results indicated that the soluble expression of PLA1 was at low level, which was possibly due to the toxicity of PLA1 to the host. In contrast, the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium. Furthermore, the renaturation of PLA1 was achieved through a direct dilution method, yielding 29.6 U/mL PLA1 activity after 16 h of renaturation at 4 °C. For improving the efficiency of the dilution refolding process, a continuous refolding strategy was established, and 155 U/mL PLA1 activity was derived from the continuous refolding process. With soybean lecithin as the substrate, the specific activity of purified renatured PLA1 was 1380 U/mg and the optimal temperature and pH was found to be 60 °C and 6.5. In addition, the renatured PLA1 was observed with better activity towards phosphatidyl inositol, whilst lipase activity was detected when the catalyzing temperature was below 55 °C. Overall, this study provides a possible solution to obtain calcium-independent PLA1 with high yield by heterologous expression in E. coli and hence to promote its further application in the field of food industry.

Development of a two-step refolding method for reteplase, a rich disulfide-bonded protein

Abstract Correct formation of disulfide bonds is crucial in the refolding of multi-disulfide-bonded inclusion body proteins. Covalent aggregation of proteins due to incorrect cross-linking of disulfide bonds decreases refolding yield. This study presents a two-step refolding method for increasing refolding yield of disulfide-bonded proteins. In this method disulfide bond formation is prevented in the first step of refolding, then in second step disulfide exchange reactions are promoted by gradual increasing of pH and cysteine concentration in refolding buffer. Recombinant tissue plasminogen activator (r-TPA, reteplase) with nine disulfide bonds was used as the model protein. Refolding yield of reteplase with two-step refolding method improved by 2.1-fold compared to dilution refolding. The successful application of two-step strategy in improving refolding yield of reteplase reveals the efficacy of this strategy for decreasing aggregation and improving correct refolding of inclusion body proteins.

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics.

High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration

Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5–4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity.

Optimization of dilution refolding conditions for a camelid single domain antibody against human beta-2-microglobulin

Single domain antibody (sdAb) is often expressed as inclusion bodies in Escherichia coli cytoplasm. Establishing an effective in vitro refolding method for sdAb obtained from inclusion bodies would be important for sdAb research. In this study, dilution refolding condition for a camelid sdAb specific against human beta-2-microglobulin was optimized for the sdAb purified from the inclusion bodies of E. coli BL21 (DE3). Single factor methods based on protein concentration, velocity of dilution, incubation time and refolding buffer composition were first investigated. Then the key refolding buffer compositions were selected for further optimization by means of the Box–Behnken design of response surface methodology (RSM). The activity of the refolded sdAb was determined by measuring its specific antigen-binding ability using indirect ELISA. The optimized refolding condition of sdAb consisted of a 10-fold dilution in 10mM Tris–HCl (pH 8.0) containing 1.24mM GSH, 1mM GSSG, 352mM l-Arg, 0.65% PEG-2000, and a 16h incubation at 4°C. Further comparison of the activities between the refolded sdAb and purified soluble sdAb expressed in E. coli Rosetta-gami (DE3) pLysS indicated that the sdAb was correctly refolded, as assayed by isothermal titration calorimetry. This work could provide an important strategy for the recombinant production and application of sdAb.

Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VH–VL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.

Multi-variable operational characteristic studies of on-column oxidative protein refolding at high loading concentrations

Chromatographic-based protein refolding techniques have proven to be superior to conventional dilution refolding methods, due to the higher loading concentration and simultaneous purification. Among these techniques, Size Exclusion Chromatography (SEC) has in particular been demonstrated as an effective method for refolding of variety of proteins. To date existing studies of protein refolding at high concentrations (>1mg/mL) in SEC have primarily been conducted as single factor studies, in which a single parameter is varied to assess impact on operating performance, which does not allow for determination of the interactions of different operating parameters and optimized operating conditions. In this work a multi-variable investigation of size exclusion protein refolding at high protein concentration using lysozyme as a model protein was performed, in order to quantify the interaction of factors and optimize performance. It was observed when l-arginine is used as an additive the refolding yield becomes independent of the protein concentration and refolding buffer pH, providing that a redox couple is used to assist the reformation of disulfide bridges. Furthermore, the pore accessibility for small molecules was reduced at the presence of this additive particularly at higher protein concentrations indicating slower removal of these molecules and a possible additional mechanism of aggregation prevention. Using the subsequent optimized refolding buffer, a refolding yield of more than 90% was obtained for up to 40mg/mL loading concentration of lysozyme which has only been reported for a urea gradient SEC (8–2M) with lower equilibration and elution flow rates due to high viscosity of buffer containing high concentrations of urea.

One-step refolding and purification of recombinant human tumor necrosis factor-α (rhTNF-α) using ion-exchange chromatography.

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic-based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor-α (rhTNF-α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF-α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF-α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10(8) IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF-α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF-α refolding in terms of specific bioactivity and mass recovery.

Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling

An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from Npro fusion pep6His and Npro fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes – one using urea for IB dissolution, the other one using NaOH for IB dissolution – and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes.

Refolding of recombinant human interferon gamma inclusion bodies in vitro assisted by colloidal thermo-sensitive poly(N-isopropylacrylamide) brushes grafted onto the surface of uniform polystyrene cores

Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γin vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55nm and 110nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33°C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15°C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro.

An Automatic Refolding Apparatus for Preparative-Scale Protein Production

Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.

Highly neurotoxic monomeric α-helical prion protein

Prion diseases are infectious and belong to the group of protein misfolding neurodegenerative diseases. In these diseases, neuronal dysfunction and death are caused by the neuronal toxicity of a particular misfolded form of their cognate protein. The ability to specifically target the toxic protein conformer or the neuronal death pathway would provide powerful therapeutic approaches to these diseases. The neurotoxic forms of the prion protein (PrP) have yet to be defined but there is evidence suggesting that at least some of them differ from infectious PrP (PrP(Sc)). Herein, without making an assumption about size or conformation, we searched for toxic forms of recombinant PrP after dilution refolding, size fractionation, and systematic biological testing of all fractions. We found that the PrP species most neurotoxic in vitro and in vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form of PrP. TPrP caused autophagy, apoptosis, and a molecular signature remarkably similar to that observed in the brains of prion-infected animals. Interestingly, highly α-helical intermediates have been described for other amyloidogenic proteins but their biological significance remains to be established. We provide unique experimental evidence that a monomeric α-helical form of an amyloidogenic protein represents a cytotoxic species. Although toxic PrP has yet to be purified from prion-infected brains, TPrP might be the equivalent of one highly neurotoxic PrP species generated during prion replication. Because TPrP is a misfolded, highly neurotoxic form of PrP reproducing several features of prion-induced neuronal death, it constitutes a useful model to study PrP-induced neurodegenerative mechanisms.

Characterization of a novel lipase and its specific foldase from Acinetobacter sp. XMZ-26

A novel operon containing a lipase gene (lip26) and its specific foldase gene (lif26) was discovered from Acinetobacter sp. XMZ-26 by creating and screening a gene library and then using genome walking. The amino acid sequence of Lip26 and Lif26 showed only 46.4% and 37.3% identity with the LipA and LipB (Lif) sequences from Acinetobacter sp. SY-01, respectively. The expressed recombinant Lip26 formed inactive inclusion bodies in Escherichia coli. However, the active Lip26 was refolded by the dilution refolding method with the assistance of purified recombinant Lif26, and the refolded Lip26 had a high specific activity. Lip26 hydrolyzed p-nitrophenyl (pNP) esters of fatty acids with C2 to C16 acyl chain lengths and had a substrate preference for pNP myristate. Maximal Lip26 activity was dependent on both the temperature (55°C) and pH (9.0). In addition, Lip26 was capable of maintaining its activity in the presence of many detergents and organic solutions, and its activity was enhanced by the presence of Ca2+, Mn2+, and Ba2+. To directly obtain active Lip26, an E. coli strain was co-transformed with two expression plasmids containing the lip26 and lif26 genes. The co-expression of both proteins in vivo resulted in the expression of half of the recombinant Lip26 as a soluble protein with demonstrable lipase activity. A direct protein interaction between Lif26 and Lip26A was detected by both a pull-down assay and a yeast two-hybrid experiment.

High productivity chromatography refolding process for Hepatitis B Virus X (HBx) protein guided by statistical design of experiment studies

The Hepatitis B Virus X (HBx) protein is a potential therapeutic target for the treatment of hepatocellular carcinoma. However, consistent expression of the protein as insoluble inclusion bodies in bacteria host systems has largely hindered HBx manufacturing via economical biosynthesis routes, thereby impeding the development of anti-HBx therapeutic strategies. To eliminate this roadblock, this work reports the development of the first ‘chromatography refolding’-based bioprocess for HBx using immobilised metal affinity chromatography (IMAC). This process enabled production of HBx at quantities and purity that facilitate their direct use in structural and molecular characterization studies. In line with the principles of quality by design (QbD), we used a statistical design of experiments (DoE) methodology to design the optimum process which delivered bioactive HBx at a productivity of 0.21 mg/ml/h at a refolding yield of 54% (at 10 mg/ml refolding concentration), which was 4.4-fold higher than that achieved in dilution refolding. The systematic DoE methodology adopted for this study enabled us to obtain important insights into the effect of different bioprocess parameters like the effect of buffer exchange gradients on HBx productivity and quality. Such a bioprocess design approach can play a pivotal role in developing intensified processes for other novel proteins, and hence helping to resolve validation and speed-to-market challenges faced by the biopharmaceutical industry today.

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM L-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of L-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that L-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.

Efficient refolding of a hydrophobic protein with multiple S–S bonds by on-resin immobilized metal affinity chromatography

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S–S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.

Characterization of MHC/peptide complexes refolded by a one-step ion-exchange chromatography

The current refolding process of MHC/peptide complexes is low-yield and time-consuming, thereby limiting the wide uses of MHC/peptide multimers. Here the heavy chain protein of MHC/peptide complex (H-2K b/TRP2 180–188) was immobilized onto an ion-exchange chromatography column, and the β2m or TRP2 180–188-fused β2m protein, which renatured previously in refolding buffer, was able to pass through the column for the gradient refolding. This strategy refolds, concentrates and purifies MHC/peptide complexes in a single integrated step, achieving a high level of process simplification and automation. Using this on-column refolding method, MHC/peptide complexes could be prepared within 24 h with a refolding yield of over 20%. Anti-H-2K b mAb staining and flow cytometric analyses revealed that the on-column refolded H-2K b/TRP2 180–188 complexes had conformational characteristics similar to the dilution refolded H-2K b/TRP2 180–188 complexes and the commercial ones. Furthermore, H-2K b/TRP2 180–188 tetramer staining and the enumeration of TRP2 180–188-specific T cells and H-2K b-alloreactive T cells confirmed that the H-2K b/TRP2 180–188 complexes prepared by on-column refolding or dilution refolding had comparable TCR-binding ability. These data demonstrate a novel, simple and efficient refolding strategy for the generation of MHC class I/peptide complexes.

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.

Size-exclusion chromatographic protein refolding: Fundamentals, modeling and operation

Size-exclusion chromatography (SEC) has proven its capability to refold a variety of proteins using a range of gel filtration column materials, demonstrated in the growing body of experimental evidence. However, little effort has been allocated to the development of mechanistic models describing size-exclusion chromatographic refolding reactors (SECRR). Mechanistic models are important since they provide a link between process variables like denatured and reduced protein feed concentration ( C f,D&R), flow rate, column length, etc., and performance indicators like refolding yield ( Y N), thereby opening the possibility for in silico design of SECRRs. A critical step, in the formulation of such models, is the selection of an adequate reaction mechanism, which provides the direct link between the separation and the refolding yield. Therefore, in this work we present a methodology using a SEC refolding reactor model, supported by a library of reaction mechanisms, to estimate a suitable reaction scheme using experimental SEC refolding data. SEC refolding data is used since it provides information about the mass distribution of monomers and aggregates after refolding, information not readily available from batch dilution refolding data alone. Additionally, this work presents (1) a systematic analysis of the reaction mechanisms considered using characteristic time analysis and Damköhler maps, revealing (a) the direct effect of a given reaction mechanism on the shape of the SEC refolding chromatogram (number of peaks and resolution) and (b) the effect that the competition between convection, refolding and aggregation is likely to have on the SEC refolding yield; (2) a comparison between the SECR reactor and the batch dilution refolding reactor based on mechanistic modeling, quantitatively showing the advantages of the former over the latter; and (3) the successful application of the modeling based strategy to study the SEC refolding data of an industrially relevant protein. In principle, the presented modeling strategy can be applied to any protein refolded using any gel filtration material, providing the proper mass balances and activity measurements are available.

A novel protein refolding system using lauroyl- l-glutamate as a solubilizing detergent and arginine as a folding assisting agent

More than 50 detergents, including acylated amino acid derivatives, were screened for their ability to solubilize and refold recombinant proteins expressed as inclusion bodies. Two model proteins, human interleukin-6 and microbial transglutaminase, were solubilized by these detergents and the solubilized proteins were rapidly diluted for testing their solubilization and refolding effectiveness. Long chain-acylated amino acid derivatives having dicarboxylic acid moieties were found to be superior to others under the conditions tested. In particular, lauroyl- l-glutamate (C12- l-Glu) showed the highest recovery of the native proteins. The effectiveness of dilution refolding was greatly improved by adding aggregation suppressive arginine into the refolding solvents. To gain understanding how this detergent works, interactions between detergents and proteins were examined using spectroscopic and native gel electrophoretic analyses, showing ideal properties for C12- l-Glu as a solubilzing agent, i.e. highly reversible nature of the detergent binding to the model globular proteins and of the conformational changes. These properties most likely have contributed to the effective protein solubilzation and refolding of inclusion bodies using C12- l-Glu and arginine.