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.

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.

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 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.

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.

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.

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, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and 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 and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and 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.

Refolding of OPTA-labeled creatine kinase denatured by guanidinium chloride

Although refolding of fully denatured protein molecules in vitro is not a valid model of folding of the nascent peptide chain in a biologically active protein in vivo, it can provide some useful information for protein folding. Therefore, refolding of denatured proteins has been extensively studied in recent years. It has been previously reported that during the refolding of guanidine or urea denatured creatine kinase, the enzymatic activity and the na-

Effects of Glycerol in the Refolding and Unfolding of Creatine Kinase

The effects of glycerol in the refolding, reactivation, unfolding, and inactivation of guanidine denatured creatine kinase were studied by observing the fluorescence emission spectra and the circular dichroism spectra, and by recovery and inactivation of enzymatic activity and aggregation. The results show that low concentrations of glycerol (<25%) improve the refolding yields of creatine kinase, but high glycerol concentrations decrease its recovery. Glycerol favors the secondary structural formation and inhibits aggregation of creatine kinase as proline does. These systematic observations further support the suggestion that low concentrations of glycerol possibly play a chaperone role in the refolding of creatine kinase. In addition, glycerol reduces the inactivation and unfolding rate of creatine kinase, increases the change in transition free energy of unfolding (ΔΔG u) and stabilizes its active conformation relative to the partially unfolded state with no glycerol. In the presence of glycerol, the inactivation and unfolding dynamics of creatine kinase are related to glycerol concentrations. Glycerol blocks the exposure of hydrophobic areas and the dissociation of dimers, and protects creatine kinase against guanidine denaturation in a concentration dependent manner. This study suggests that glycerol as an energy substrate for metabolism and organic components in vivo, assists correct protein folding, maintains adequate rates of enzymatic catalysis and stabilizes the protein secondary and tertiary conformations.

Studies on the Refolding of Egg White Lysozyme Denatured by Urea Using ＂Phase Diagram＂ Method of Fluorescence

The refolding of reduced and non-reducing egg white lysozymes in a urea solution was studied by a ＂phase diagram＂ method of fluorescence. The result showed that in the refolding of the reduced egg white lysozyme, an intermediate state of an egg white lysozyme exists at the urea concentrations in a final renaturation solution being about 4.5 mol/L, their refolding follows a three-state model; while in the refolding of the non-reducing egg white lysozyme, two intermediate states exist at the urea concentrations being separately 4.0 and 2.5 mol/L, and their refolding follows a four-state model. Through the comparison between the unfolding and refolding of an egg white lysozyme in the urea solution, it was found that both of the refolding of reduced and non-reducing egg white lysozyme molecules was irreversible to their unfolding in the urea solution. Finally, a suggested refolding was separately presented for the reduced and non-reducing egg white lysozymes in the urea solution.

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

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

Profiling neuroprotective potential of trehalose in animal models of neurodegenerative diseases:a systematic review

Trehalose,a unique nonreducing crystalline disaccharide,is a potential disease-modifying treatment for neurodegenerative diseases associated with protein misfolding and aggregation due to aging,intrinsic mutations,or autophagy dysregulation.This systematic review summarizes the effects of trehalose on its underlying mechanisms in animal models of selected neurodegenerative disorders(tau pathology,synucleinopathy,polyglutamine tract,and motor neuron diseases).All animal studies on neurodegenerative diseases treated with trehalose published in Medline(accessed via EBSCOhost)and Scopus were considered.Of the 2259 studies screened,29 met the eligibility criteria.According to the SYstematic Review Center for Laboratory Animal Experiment(SYRCLE)risk of bias tool,we reported 22 out of 29 studies with a high risk of bias.The present findings support the purported role of trehalose in autophagic flux and protein refolding.This review identified several other lesser-known pathways,including modifying amyloid precursor protein processing,inhibition of reactive gliosis,the integrity of the blood-brain barrier,activation of growth factors,upregulation of the downstream antioxidant signaling pathway,and protection against mitochondrial defects.The absence of adverse events and improvements in the outcome parameters were observed in some studies,which supports the transition to human clinical trials.It is possible to conclude that trehalose exerts its neuroprotective effects through both direct and indirect pathways.However,heterogeneous methodologies and outcome measures across the studies rendered it impossible to derive a definitive conclusion.Translational studies on trehalose would need to clarify three important questions:1)bioavailability with oral administration,2)optimal time window to confer neuroprotective benefits,and 3)optimal dosage to confer neuroprotection.

可溶性人TRAIL分子的制备及其抗肿瘤活性

The cDNA encoding human TRAIL extracellular region(amino acids 41—281) was amplified by reverse transcription(RT) PCR from total RNA derived from human acute promyelocytic leukemia cell line HL 60. After sequencing, the cDNA was cloned into the vector pQE 80L and transformed into E.coli DH5 α . By IPTG induction, the soluble TRAIL 41—281 (sTRAIL 41—281 ) protein was expressed with the 40% of total bacteria protein. Inclusion bodies were dissolved into 8 mol/L urea, purified by Ni NTA chromatography column, the product with over 90% purity was obtained. After refolding by dialysis, the active trimer form of sTRAIL 41～281 was derived from the renatured proteins by gel filtration chromatography. The MTT assay, flow cytometry and DNA fragmentation assay showed that the refolded sTRAIL 41～281 could potently inhibit the growth of Jurkat cells and induce apoptosis, confirmed the apoptosis inducing activity of sTRAIL 41～281 on tumor cells, it will benefit the further research of TRAIL.