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.

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.

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.

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.

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.

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.

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.

枸杞LbSPL6重组蛋白的纯化和复性

SPL(Squamosa promoter binding protein-like,SPL)是植物特有的转录因子,在植物花和果实的生长发育、次生代谢、调控植物适应性等过程中起着重要作用。SPL蛋白中含有高度保守的SBP功能结构域,通过特异性结合基因的启动子元件,调控相关基因的表达。对已获得的枸杞pET28a-LbSPL6融合蛋白进行诱导表达条件的优化,并对形成的包涵体蛋白进行纯化和复性。结果表明:诱导温度37℃、c(IPTG)=1 mmol/L诱导4 h后,LbSPL6蛋白的表达量最高;原核表达的LbSPL6蛋白,在各种诱导条件下的存在形式都为包涵体;通过超声破碎回收包涵体,并将沉淀在变性缓冲液中进行溶解复性,利用Ni-NTA柱亲和层析纯化蛋白,从而获得有活性的重组LbSPL6融合蛋白。蛋白免疫检测结果进一步证实纯化后的蛋白为LbSPL6重组蛋白。试验纯化得到的重组LbSPL6蛋白为进一步深入研究枸杞LbSPL6蛋白的功能奠定了基础。

pH偏移处理对猪肝蛋白乳化特性的影响

为探究猪肝蛋白(porcine liver protein,PLP)的pH偏移诱导重折叠改性方法与机制。猪肝蛋白溶液经pH偏移处理(pH3~11)后再将pH值调整到中性,经过冻干处理后再测定改性猪肝蛋白的溶解度、乳化活性与稳定性、表面疏水性、乳液粒径与Zeta电位、活性巯基及内源荧光光谱、红外光谱。结果表明,在pH酸性偏移条件下,PLP的溶解度、乳化活性及乳化稳定性均有所下降,乳液粒径增大、Zeta电位绝对值下降,而pH碱性偏移处理会使PLP的溶解度和乳化活性、乳化稳定性提高,乳液粒径减小、Zeta电位绝对值上升;改性后PLP的荧光强度及活性巯基含量降低,表明pH偏移处理对蛋白质三级结构产生显著影响,而根据红外光谱结果显示,其对蛋白质二级结构影响较小。因此,pH碱性偏移处理可作为提高猪肝蛋白的乳化性、溶解性等功能特性的有效手段。

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 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.

Expression of Fragaria ananassa Osmotin-like Protein(FaOLP2) in E. coli:Purification and Antifungal Activity

[ Objective] The FaOLtr2 （ Frago^ia ananassa osmotin-like protein） is a functional homolog of PR5-1ike protein. This study was undertaken to produce recombinant FaOLP2 and to identify its antifungal activity. [ Method] The ORF of FaOLP2 （ accession number DQ325524） was cloned into pET22b vector to con- stroct the pET22b-FaOLP2 plasmid. The recombinant mature FaOLP2 was expressed in E. coli Rosetta-gami B （DE3） by inducing with I nunol/L IPTG and found exclusively in insoluble inclusion bodies. As FaOLP2 requires the correct formation of eight disulfide bonds, but there were no obvious effect to correctly form these by expression at different temperatures and high osmotic pressure （ supplemented Betaineand and D-Sorbitol）, we used an in vitro method to refold E. coli expressed FaOLP2 by gradually elution using reduced：oxidized gluthatione redox buffer, followed by 8 mol/L urea solubilized His6-tagged mature FaOLP2 protein, which was affinity-purified by an immobilized-metal （Ni2＋ ） affinity chromatography （IMAC） column. [ Result] This method generated biologically active conformations of the recombinant mature FaOLP2 that displayed antifungal activity against Ustilaginoides virens, a plant pathogenic fungus, which causes rice false smut. [ Conclusion] This study laid the foundation for further biotechnological application of the novel protein.

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.

大肠杆菌表达包涵体蛋白体外复性研究进展

包涵体形成是大肠杆菌表达重组蛋白常见的问题。因此,为获得大量的活性蛋白而对其包涵体进行复性的研究引起了人们极大兴趣。本文综述了近年来发展起来的低分子量添加物、反胶团、分子伴侣、层析复性等新的复性方法、复性检测方法和体外复性机制。

SA-TNF-α融合蛋白高效表达、纯化及复性研究

目的研究链亲和素标记的人肿瘤坏死因子α(SA-TNF-α)融合蛋白的纯化、复性方法,并研究其生物学功能。方法在大肠杆菌中表达SA-TNF-α融合蛋白,对表达的SA-TNF-α融合蛋白采用镍金属螯合(Ni-NTA)层析柱进行纯化,分别在尿素体系和盐酸胍体系中复性,Western blot对其进行鉴定。MTT法检测SA-TNF-α融合蛋白对L929细胞的杀伤活性,流式细胞仪分析SA-TNF-α融合蛋白对生物素化的MB49细胞锚定修饰率。结果SA-TNF-α融合蛋白在大肠杆菌中实现了高效表达,表达的目标蛋白占菌体蛋白30%以上,镍金属螯合(Ni-NTA)层析柱纯化的SA-TNF-α融合蛋白纯度达到95.7%,经过两种体系复性形成的SA-TNF-α融合蛋白的二聚体和多聚体均具有双功能活性:既对L929细胞有杀伤活性又能锚定修饰生物素化的MB49细胞,锚定修饰率大于90%。结论初步建立了SA-TNF-α融合蛋白制备工艺,SA-TNF-α二聚体和多聚体均具有双功能活性,SA-TNF-α融合蛋白的研制有望为肿瘤的治疗提供新的治疗方法及新型药物。

猪瘟病毒E2蛋白在大肠杆菌中的表达及其可溶性分析

旨在表达出囊膜糖蛋白E2,为猪瘟病毒的亚单位标记疫苗研制、血清学诊断等提供依据。以pMD-18T-E2为模板,扩增猪瘟病毒E2基因,克隆至原核表达载体pET-28a,构建重组原核表达质粒pET-28a-E2,并在大肠杆菌Rosetta(DE3)中诱导表达。结果显示,通过SDS-PAGE凝胶电泳可检测到46ku的蛋白条带,与预期结果一致;目的蛋白主要以包涵体形式存在。将E2蛋白变性纯化后,分别采用透析复性和稀释复性2种方法进行复性,成功得到可溶性E2蛋白。

高效弱阳离子交换色谱法对脲还原变性溶菌酶的折叠研究

用高效弱阳离子交换色谱(HPWCX)对脲还原变性溶菌酶(Lys)进行了复性研究.在流动相中脲浓度固定为4.0mol?L-1和选用对天然态蛋白有稳定作用的硫酸铵为盐或置换剂时,在蛋白浓度为15.0～50.0mg?mL-1时,HPWCX法比稀释法活性回收率高.为了提高Lys的质量及活性回收率对所用色谱条件进行了优化研究,当蛋白起始浓度为20.0mg?mL-1时,Lys的质量回收率和活性收率分别为97.8%和95.4%.表明此种方法简便且有可能对其他还原变性蛋白的复性具有通用性.

胍变及脲变α-淀粉酶的研究 Ⅰ.用高效疏水色谱法研究变性机理和复性效率

用疏水性强弱不同的两种色谱柱对７．０ｍｏｌ／Ｌ盐酸胍及８．０ｍｏｌ／Ｌ脲变性的α－淀粉酶变体和在疏水色谱介质表面上折叠的中间体进行了分离和复性。通过研究和比较发现，两者的变性机理和形成折叠中间体的个数以及复性效率均不相同。在用疏水性较弱的疏水色谱柱对脲变α－淀粉酶的折叠中间体进行分离时，得到了疏水性接近连续的、数目很多的中间体。用疏水性较强的疏水色谱柱对胍变α－淀粉酶进行复性的效果较好。还研究了柱温变化对其折叠、分离效果和复性效率的影响。

使用三态模型选择基因重组蛋白质的复性条件

论述了使用蛋白质复性过程中的三态模型来选择基因重组蛋白质的复性条件，并对以往使用的复性条件和新的复性方法进行了合理的解释，为从理论上选择蛋白质的复性条件提供了依据。