采用不同疏水相互作用色谱柱从包涵体中快速制备重组人干细胞因子

为了提高重组人干细胞因子(rhSCF)的复性效率,改进了高效疏水相互作用色谱(HPHIC)纯化和复性rhSCF的方法。首先将目标蛋白溶解于8.0 mol/L脲中,然后将rhSCF包涵体的提取液直接进样到不同规格的HPHIC柱进行纯化和复性。优化了固定相配基结构和流动相组成等实验条件,结果表明,本方法可以快速地获得高质量回收率和高生物活性的rhSCF,rhSCF在40 min内即可完成复性与纯化,目标蛋白的纯度在95.5%以上,质量回收率高于49.6%。通过体积排阻色谱和基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)的分析,确认rhSCF以单体存在。结果进一步证明HPHIC法是同时复性和纯化重组蛋白的有效工具。     

还原变性核糖核酸酶在疏水性液-固界面上的复性

采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。     

HPHIC固定相对脲变α-糜蛋白酶复性的影响

依据计量置换保留理论所得到的参数lgI, 来测定不同构象态α-糜蛋白酶(α-Chy)在两种不同高效疏水相互作用色谱(HPHIC)固定相表面的折叠自由能, 发现脲变α-Chy在HPHIC固定相表面获取的折叠自由能比溶液中的高很多, 不同HPHIC固定相表面为脲变α-Chy提供不同的折叠自由能, 且都随变性剂脲浓度的增大而增大;通过对不同HPHIC色谱柱后复性α-Chy的比活测定, 还发现脲变α-Chy的复性效率与其从固定相表面的折叠自由能有关, 同一构象的α-Chy从固定相表面得到的折叠自由能越高越有利于其折叠成天然蛋白质.     

蛋白折叠液相色谱法中流动相对重组人干扰素-γ质量回收率的影响

蛋白折叠液相色谱法(PFLC)用于变性蛋白质复性并同时纯化时对流动相组成及其洗脱条件的要求远较通常的液相色谱法高。用端基为PEG-200的高效疏水作用色谱固定相对重组人干扰素-γ(rhIFN-γ)进行纯化并同时复性,详细研究了流动相组成、梯度洗脱模式和流速对rhIFN-γ质量回收率和活性的影响。分别以3.0 mol/L (NH4)2SO4 +0.05 mol/L KH2PO4(pH 7.0)和0.05 mol/L KH2PO4(pH 7.0)为流动相A和B,采用35 min非线性梯度洗脱时,所得rhIFN-γ的质量回收率最高。     

敬钊毒素-V剪切体Y1-JZTX-V的化学合成与氧化复性及其钠通道活性鉴定

应用芴甲氧羰基(Fmoc)固相方法化学合成了敬钊毒素-V（JZTX-V）分子N-端酪氨酸残基剪切体(Y1-JZTX-V),并且通过反相高效液相色谱和质谱对不同条件下的氧化复性结果进行监测,从而得到该剪切体的最佳氧化复性条件:0.1 mol/L Tris-HCl缓冲液、pH 7.50、1 mmol/L还原型谷胱甘肽(GSH)、0.1 mmol/L氧化型谷胱甘肽(GSSG)、样品浓度为0.05 mg/L、复性温度为4 ℃。膜片钳电生理实验结果显示敬钊毒素-V剪切体Y1-JZTX-V对大鼠背根神经节(DRG)细胞上表达的河豚毒素不敏感型(TTX-R)与河豚毒素敏感型(TTX-S)钠电流均有抑制作用,其半数抑制浓度(IC50)分别为(160±2.5) nmol/L和(39.6±3.2) nmol/L。与天然的敬钊毒素-V相比,该剪切体对大鼠DRG细胞上的TTX-S钠电流的抑制作用基本一致,但对TTX-R钠电流的抑制作用却大大降低,表明敬钊毒素-V分子N-端的酪氨酸残基是一个与TTX-R钠通道结合活性相关的氨基酸残基。     

海南捕鸟蛛毒素-Ⅲ的固相化学合成和氧化复性

应用芴甲氧羰基(Fmoc)固相化学合成了海南捕鸟蛛毒素-Ⅲ,并优化了其最佳氧化复性条件。最佳的氧化复性条件为pH 7.5的重蒸水溶液体系,样品质量浓度为0.1 g/L,还原型谷胱甘肽和氧化型谷胱甘肽的浓度分别为 1.0 mmol/L和0.1 mmol/L、L-Arg浓度为1.0 mol/L。复性产物经质谱测定其相对分子质量为3607.68;与天然毒素等量混合后用高效液相色谱分析得到单一峰;膈神经-膈肌标本生理实验结果表明,合成的毒素具有与天然毒素相同的生物学活性,从而可确定二者在结构与功能上具有一致性。     

脲变性牛碳酸酐酶B的稀释复性过程及其集聚作用研究

采用非变性聚丙烯酰胺凝胶电泳、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效凝胶排阻色谱以及激光光散射光谱研究了脲变性牛碳酸酐酶B的稀释复性过程及其集聚作用。在脲变性牛碳酸酐酶B的稀释复性过程中,当最终复性液中脲浓度大于2.0mol/L时,牛碳酸酐酶B在复性液中以单分子和二分子集聚体形式存在;当最终复性液中脲浓度小于2.0mol/L大于1.0mol/L时,牛碳酸酐酶B在复性液中以单分子、二分子集聚体和少量多分子集聚体形式存在;而当最终复性液中脲浓度小于等于1.0mol/L时,脲变性牛碳酸酐酶B复性时会形成均匀透明的上清和不透明的沉淀,牛碳酸酐酶B在上清和沉淀中达到动态解离平衡,且在两相中都以单分子、二分子集聚体和少量多分子集聚体形式存在。溶液中二分子和多分子牛碳酸酐酶B集聚体是通过牛碳酸酐酶B分子之间的疏水和静电相互作用力而形成的,当溶液中这些成分达到一定浓度并且溶液中脲的浓度小于某一个值时,它们之间会通过非共价形式形成沉淀。     

重组羧肽酶原B的体外变复性研究

重组羧肽酶原B在大肠杆菌中过量表达时形成包涵体，需要经过体外交复性后才能获得生物活性．为了提高羧肽酶原B的复性效率，首先对包涵体的溶解条件进行了优化．对比了极端pH条件、各种变性剂和一些表面活性剂对包涵体的溶解效果；并且在较弱的碱性条件下得到了很好的包涵体溶解效果．接着，通过对缓冲液中蛋白浓度、PH值、温度、氧化还原对（GSH：GSSG）比值的定量分析，确定了复性液的基本成分；比较了不同浓度的尿素对防止聚集、提高复性效率的影响；另外还对比了加样方式的影响，最终确定了重组羧肽酶原B体外复性的最佳条件，即20mmol／L Tris—HCl，pH=9．5，150μg／mL Pro—CPB concentration，1mmol／L GSH，0．5mol／L urea．复性效率比最初提高了3倍左右．     

用疏水色谱对还原型胍变性牛胰岛素的折叠特性研究

用疏水相互色谱(HPHIC)对还原胍变性牛胰岛素在疏水界面上的折叠与复性进行了研究.结果表明,采用普通流动相时,对还原胍变胰岛素的复性效果较差,而采用氧化型流动相可使其复性效率提高到66%,并用反相色谱(RPLC)、紫外吸收光谱、荧光光谱及MALDI-TOF对其复性效果进行了验证.同时与体积排阻色谱(SEC)和稀释法对还原胍变胰岛素的复性结果进行了比较.结果表明,SEC根本无法使还原胍变胰岛素复性,而稀释法的复性效率仅有2%.这进一步表明HPHIC是变性蛋白复性的有效工具,变性蛋白在疏水界面折叠过程中,蛋白质与固定相之间的疏水相互作用对蛋白折叠起着关键性的作用,是蛋白折叠的主要驱动力.     

高效疏水作用色谱填料的合成及其在重组人干扰素纯化中的应用

高效疏水作用色谱（HPHIC）是利用不同蛋白质表面疏水区域与填料之间具有不同疏水作用进行分离的．由于HPHIC采用盐水体系作为流动相，配体采用极性的有机基团，使蛋白质可以在十分温和的条件下进行分离，且保持其生物活性基本不变[1，2]．自80年代中期以来，HPHIC在蛋白质的分离纯化上得到了广泛的应用．在90年代初期，随着基因工程技术的发展，HPHIC同时也被应用到基因工程的下游纯化技术上[3，4]．本文中我们合成了一OCH2CH3为端基的填料，检验了该填料的分离效果，并利用该填料对酵母菌表达的人αA-干扰素、大肠杆菌（E．col…     

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.     

Solubilization and Refolding with Simultaneous Purification of Recombinant Human Stem Cell Factor

Recombinant human stem cell factor (rhSCF) was solubilized and renatured from inclusion bodies expressed in Escherichia coli. The effect of both pH and urea on the solubilization of rhSCF inclusion bodies was investigated; the results indicate that the solubilization of rhSCF inclusion bodies was significantly influenced by the pH of the solution employed, and low concentration of urea can drastically improve the solubilization of rhSCF when solubilized by high pH solution. The solubilized rhSCF can be easily refolded with simultaneous purification by ion exchange chromatography (IEC), with a specific activity of 7.8 × 10⁵ IU·mg⁻¹, a purity of 96.3%, and a mass recovery of 43.0%. The presented experimental results show that rhSCF solubilized by high pH solution containing low concentration of urea is easier to be renatured than that solubilized by high concentration of urea, and the IEC refolding method was more efficient than dilution refolding and dialysis refolding for rhSCF. It may have a great potential for large-scale production of rhSCF.     

疏水作用色谱法同时纯化及复性基因重组人干扰素-α

 使用高效疏水作用色谱直接从大肠杆菌表达的基因重组人干扰素 α(rhIFN α)包涵体的裂解液中纯化了rhIFN α ,并在纯化的同时获得了高的复性效率 ,使复性和纯化一步完成 ,大大地简化了操作步骤。用凝胶排阻色谱对该法纯化的rhIFN α进行了纯度测定 ,纯度达到 95 %以上。该法的活性回收率分别比稀释法和透析法高 1 0倍和 1 6倍。     

Urea-induced Inactivation and Unfolding of Recombinant Phospholipid Hydroperoxide Glutathione Peroxidase from Oryza sativa

Phospholipid hydroperoxide glutathione peroxidase is an antioxidant enzyme that has the highest capability of reducing membrane-bound hydroperoxy lipids as compared to free organic and inorganic hydroperoxides amongst the glutathione peroxidases.In this study,urea-induced effects on the inactivation and unfolding of a recombinant phospholipid hydroperoxide glutathione peroxidase(PHGPx)from Oryza sativa were investigated by means of circular dichroism and fluorescence spectroscopy.With the increase of urea concentration,the residual activity of OsPHGPx decreases correspondingly.When the urea concentration is above 5.0 mol/L,there was no residual activity.In addition,the observed changes in intrinsic tryptophan fluorescence,the binding of the hydrophobic fluorescence probe ANS,and the far UV CD describe a common dependence on the concentration of urea suggesting that the conformational features of the native OsPHGPx are lost in a highly cooperative single transition.The unfolding process comprises of three zones:the native base-line zone between 0 and 2.5 mol/L urea,the transition zone between 2.5 and 5.5 mol/L urea,and the denatured base-line zone above 5.5 mol/L urea.The transition zone has a midpoint at about 4.0 mol/L urea.     

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。     

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Protein‐folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high‐performance size‐exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low‐urea gradient‐elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS–18% PAGE, Matrix‐Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI‐TOF‐MS) and the biological activity assay by HP‐RPLC. Copyright © 2014 John Wiley & Sons, Ltd.     

Role of stimuli-sensitive polymers in protein refolding: alpha-amylase and CcdB (controller of cell division or death B) as model proteins

Alginate, a calcium-sensitive polymer, could carry out simultaneous purification and refolding of 8 M urea/100 mM dithiothreitol (DTT) denatured and thermally denatured alpha-amylase present in a commercial preparation. Activity recoveries of 80 and 70% in the former and the latter cases, respectively, were obtained. The fluorescence spectra showed refolding, and PAGE showed the absence of any aggregates in the refolded preparation. As another example, Eudragit S-100, a pH-sensitive poly(methyl methacrylate), was used to refold CcdB (controller of cell division or death B) protein. Initial experiments with wild-type (WT) CcdB showed that Eudragit bound and precipitated (upon lowering the pH to 4.0) CcdB quantitatively from the latter's aqueous solution. The bioconjugate showed DNA gyrase inhibition activity of CcdB and could be recycled. The inclusion bodies of CcdB mutant CcdB-17P were solubilized in 8 M urea/100 mM dithiothreitol. This preparation could be refolded by precipitation with Eudragit. The fluorescence and CD spectra showed that protein refolding has occurred.     

金纳米粒子富集-高效液相色谱同时测定人血浆中三种氨基硫醇

建立了金纳米粒子富集-高效液相色谱-紫外检测(HPLC-UVD)同时测定人血浆中3种氨基硫醇(半胱氨酸(Cys)、高半胱氨酸(Hcys)、谷胱甘肽(GSH))的新方法。以Tween 20修饰的金纳米粒子作为选择探针萃取富集氨基硫醇。经二硫苏糖醇脱附后,采用SpursilTM C18柱(250 mm×4.6 mm, 5 μm)分离氨基硫醇,以60 mmol/L磷酸盐缓冲溶液(pH 2.0)等度洗脱,检测波长为200 nm。3种氨基硫醇的浓度分别在0.025～350 μmol/L、0.02～60 μmol/L、0.01～50 μmol/L内与峰面积具有良好的线性关系,相关系数均高于0.99。方法检出限(信噪比为3)分别为5.0、6.0和2.5 nmol/L,回收率为92.8%～106.0%。该方法能显著降低血浆样品中内源性物质的干扰,提高HPLC-UVD的选择性和灵敏度。将该方法应用于心血管病人血浆中上述氨基硫醇的分离测定,结果显示: 与对照组相比,疾病组血浆中的Hcys和GSH水平存在显著性差异,Cys不存在显著性差异。     

MMP-12 蛋白包涵体复性时折叠状态核磁共振图谱与荧光光谱研究

MMP-12 是癌症治疗药物靶标. 为了更好研制新药, 需要大量制备MMP-12, 但MMP-12 在大肠杆菌中以包涵体形式表达. 因此如何优化蛋白复性过程是大量获取MMP-12 蛋白的关键. 采用核磁共振、稳态荧光法、外源性ANS (8-anilinol-naphthalenesulfonic acid)荧光探针三种方法监控MMP-12 变性蛋白的再折叠过程, 以探究其复性折叠机制. 研究发现MMP-12 再折叠中点值与对应的尿素浓度几乎相等(C_m≈4, mid-point of transition). 不同尿素浓度中MMP-12 的二维¹H-¹⁵N HSQC (heteronuclear single quantum correlation)谱图显示, 尿素浓度从4 mol/L 降低到3 mol/L 是MMP-12 蛋白复性折叠的关键步骤. 据此我们将MMP-12 蛋白复性从常规的梯度透析复性方法改进成等容透析复性法(即确保尿素从4 mol/L 到3 mol/L 的浓度变化缓慢), 实现复性收率提高一倍.