New Development of Reverse Micelles and Applications in Protein Separation and Refolding

Reverse micelles bring mild and effective microenvironments in organic solvent that contain bitmolecules, which have attracted immense attention for application in the isolation of proteins, protein refolding, and enzymatic reaction. In this review, the application of reverse micelles for protein separation and refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, non- ionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential application of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for protein separation and refolding in the downstream processing of biotechnology has been pointed out.

Lysozyme refolding at high concentration by dilution and size—exclusion chromatography

This study of renaturation by dilution and size exclusion chromatogra phy (SEC) addition of urea to improve yield as well as the initial and final pro tein concentrations showed that although urea decreased the rate of lysozyme ref o lding, it could suppress protein aggregation to sustain the pathway of correct r efolding at high protein concentration; and that there existed an optimum urea c oncentration in renaturation buffer. Under the above conditions, lysozyme was su ccessfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. τ>2(t_R2 -t_R1 ), the efficiency was increased by 25% and the renaturation buffe r could be recycled for SEC refolding in continuous operation of downstream proc ess.

A Temperature-sensitive Hydrogel Refolding System: Preparation of Poly(N-isopropyl acrylamide) and Its Application in Lysozyme Refolding

Temperature-sensitive hydrogel—poly(N-isopropyl acrylamide) (PNIPA) was prepared and applied to protein refolding. PNIPA gel disks and gel particles were synthesized by the solution polymerization and inverse suspension polymerization respectively. The swelling kinetics of the gels was also studied. With these prepared PNIPA gels, the model protein lysozyme was renatured. Within 24h, PNIPA gel disks improved the yield of lysozyme activity by 49.3% from 3375.2U·mg^-1 to 5038.8U·mg^-1. With the addition of faster response PNIPA gel beads, the total lysozyme activity recovery was about 68.98% in 3h, as compared with 42.03% by simple batch dilution. The novel refolding system with PNIPA enables efficient refolding especially at high protein concentrations. Discussion about the mechanism revealed that when PNIPA gels were added into the refolding buffer, the hydrophobic interactions between denatured proteins and polymer gels could prevent the aggregation of refolding intermediates, thus enhanced the protein renaturation.

Expression, Purification, and Refolding of Recombinant Fusion Protein hIL-2/mGM-CSF

Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.

Refolding of Denatured/Reduced Lysozyme Using Weak-Cation Exchange Chromatography

Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.

pH-sensitive polymer-assisted refolding of urea-denatured fibroblast growth factor

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of FGF-2 （fibroblast growth factor-2） denatured with 8 mol/L urea and 10 mmol/L dithiothreitol at pH 7.2. The refolding of FGF-2 was performed by directly diluting denatured FGF-2 into a refolding buffer containing Eudragit S-100. The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTT method, fluorescence emission spectroscopy and reverse phase HPLC. On the other hand, the result shows the ability of Eudragit S-100 to enhance the refolding level of protein is due to the interaction between Eudragit S-100 and oositivelv charaed FGF-2.

Stability and Refolding of Prophenol Oxidase Protein with 2-Propanol in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as folded phase precursors designated as prophenol oxidase A1 and A3, and prophenol oxidase is activated with alcohol, especially 2-propanol, within a few minutes as unfolded-phase in vitro. To clarify a common effect of alcohols on proteins and peptides, the extract containing prophenol oxidase protein was prepared. Phenol oxidase activity activated with 2-propanol has been maintained stable at least 24 hours remains as it is. Protein of prophenol oxidase was not denatured opposite hypnoses known as the instability of protein with alcohol. Activated prophenol oxidase with 2-propanol remain enzyme activity with no aggregation, stable, renaturation, and the refolding phenomena occurred around the active phase within the catalytic active center of prophenol oxidase protein in Drosophila melanogaster. This study is important to induce the wide range applications of the effect in many fields for rational drag design.

Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS

The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography （WAX） coupled with MALDI-TOF MS. The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX. However, with the urea gradient elution and in the presence of GSSG/ Cyst as the ratio 1：6 in the mobile phase employed, the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the correct three disulfide bonds. The protein refolding efficiency of reduced/denatured insulin can be increased from 3 % to 34%. The effects of urea gradient and the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst, on the forming the disulfide bonds of reduced/denatured insulin were investigated in detail. The results were further tested by the separation of the WAX fraction of reduced/denatured insulin with RPLC and MALDI-TOF MS.

Refolding and Purification of Yeast Acetyl-CoA Carboxylases CT Domain Expressed as Inclusion Bodies in Escherichia coli

Acetyl-CoA carboxylase（ACCase） is a crucial enzyme in fatty acid synthesis, by regulating the first committed step in the process. Therefore, it is a potential target for the development of new compounds against obesity or as herbicides. The cDNA encoding yeast ACCase CT domains（YCTs） from Saccharvmyces cerevisiae was amplified by RT-PCR and inserted into the vector PET28a（＋） for bacterial expression of YCT fused to N-terminal His-tag（YCT-his6）. YCTs-his6 was expressed in Escherichia coli BL21（DE3） PLys as inclusion bodies, which was solubilized in 8 mol/L urea. Ni-agarose chromatography was used to purify the inclusion bodies under denaturing condition. Correct refolding was achieved via systematic dialysis to remove the denaturant gently in the presence of 0.5 mmol/L Triton X-100. The low concentration Triton X-100 was included in the refolding buffer to increase the solubilization and enhance dimeric formation of refolding proteins. The activity of the refolded YCT-his6 was 1.2 U/rag as measured in a spectrophotometric assay using malonyl-CoA as the substrate. To our knowledge, it is the first time that the bioactive YCT-his6 has been expressed successfully in E. coli and isolated from their inclusion bodies.

Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.

A New Protocol for Solubilization, Refolding and Purification of Recombinant Human Granulocyte Colony-stimulating Factor in Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.

Efficient Protein Refolding Using Surfactants at High Final Protein Concentration

The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high final refolded HEWL concentration (1 mg/mL). Hexadecyltrimethylammonium bromide (CTAB) and sucrose fatty acid monoester (DK-SS) were used to dissolve denatured HEWL without denaturants such as guanidine hydrochloride (GuHCl) and urea. When denatured HEWL was perfectly dissolved in buffer solutions containing surfactants and dithiothreitol (DTT), the concentration of CTAB was about one-twentieth times less than that of DK-SS. The concentration of CTAB strongly affected the refolding yield, and the maximum refolding yield was obtained at 0.88 mM CTAB, which is around the critical micelle concentration of CTAB. The refolding yield was influenced by the molar ratio of oxidized glutathione (GSSG) to DTT, and the maximum refolding yield was obtained when [GSSG]/[DTT] was 1.5. The refolding yield was markedly dependent upon the solution pH of HEWL, and exhibited 80% at pH 5.2.

On-column Refolding of Diphtheria Toxin Variant CRM197 by Different Metal-Chelating Affinity Chromatography Matrices

We have previously shown that the diphtheria toxin variant CRM 197 （cross-reacting material 197） can be overexpressed in Escherichia coli at high levels, yielding insoluble aggregates, which were solubilized using urea. This study reports a comparison of three matrices suitable for the purification and refolding of recombinant CRM197 by metal-chelating affinity chromatography, Moreover, we show that refolded CRM197 features enzymatic activity.

Microwave based reversible unfolding and refolding of alcohol oxidase protein probed by fluorescence and circular dichroism spectroscopy

The reversible effect of microwave mediated denaturation of protein at low exposure time of 10 s has been demonstrated for the first time. The effect of microwave (2.45 GHz and 900 W) was confirmed in a homo-octameric alcohol oxidase in aqueous solution of pH 7.5. The unfolding events did not transverse through any intermediate states and no subunits of the protein were detached during the process. The refolding of the protein achieved at 4℃ for 24 h had regenerated the native enzyme. This reversible refolding approach excludes any chemical reagent and therefore established as simple technique for protein unfolding-folding studies.

Enhanced refolding of lysozyme with imidazolium-based room temperature ionic liquids: Effect of hydrophobicity and sulfur residue

The expression of recombinant proteins in microorganism frequently leads to the formation of insoluble aggregates, inclusion bodies （IBs）. Thus, the additional in vitro protein refolding process is required to convert inactive IBs into water-soluble active proteins. This study investigated the effect of sulfur residue and hydrophobicity of imidazolium-based room temperature ionic liquids （RTILs） on the refolding of lysozyme as a model protein in the batch dilution method which is the most commonly used refolding method. When lysozyme was refolded in the refolding buffer containing [BF4]-based RTILs with a systematic variety of alkyl chain on cations varying from two to eight, less hydrophobic imidazolium cations having shorter alkyl chains were effective to facilitate lysozyme refolding. Compared to the conventional refolding buffer, 2 times higher lysozyme re- folding yield was obtained in l-ethyl-3-methylimidazolium tetrafluoroborate （[EMIM][BF4]） containing refolding buffer. The refolding yield of lysozyme was even more increased by 2.5 times when 1-butyl-3-methylimidazolium methylsulfate （[BMIM][MS]） containing sulfur residue on anion was used. The sulfur residue in [BMIM][MS] is supposed to improve the refolding yield of lysozyme which has 4 intrarnolecular disulfide bonds. For dilution-based refolding of lysozyme, the opti- mum concentrations of RTILs in refolding buffer were found to be 1.0 M [EMIM][BF4] and 0.5 M [PMIM][MS], respectively. The optimum temperate for dilution-based refolding of lysozyme with RTILs was 4 ℃.

In vitro insulin refolding:Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B,; P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates; the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain; B chain individually formed the intermediates: two A chain intermediates (P1A; P3A),; four B chain intermediates (P2B, P4B, P5B,; P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II; III, each containing two native disulfides, were formed through the recognition; interaction of transient I with P4B or P6B; the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II; III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.

CHROMATOGRAPHIC REFOLDING OF PROTEINS:MOLECULAR ACTION AND COLUMN CONTROL

Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.

Evidence for the Existence of Cross-Linked Intermediates during Unfolding and Refolding of CK in UGGE

Urea gradient gel electrophoresis (UGGE) is an important technique for studying the conformation changes of proteins during denaturation. This paper reports on an investigation of the unfolding and refolding of creatine kinase (CK) by UGGE. The native and denatured CK underwent electrophoresis in polyacrylamide gels containing a linear 08 mol/L gradient of urea perpendicular to the direction of migration. The results showed that unfolding and refolding of CK is a relatively rapid process. The denatured enzyme could refold to a conformation with activity during electrophoresis at low urea concentrations, indicating that denaturation in urea is reversible. More importantly, both the native and denatured CK were separated into multiple parallel bands through UGGE, but the bands decreased significantly when mercaptoethanol was added to the samples. The results suggest that various kinds of unfolding and refolding intermediates were formed during UGGE, which are assumed to be oligomers with disulfide bonds between peptide chains. Urea/SDS (sodium dodecylsulphate) polyacrylamide two dimensional electrophoresis proved that these unfolding and refolding intermediates formed during UGGE were oligomers which were composed of different number of subunits cross linked by disulfide bonds. The results indicate that the unfolding and refolding of CK are relatively rapid processes with some cross linked intermediates with disulfide bonds during unfolding and refolding of the enzyme.