非还原脲变性蛋白溶菌酶稀释复性过程中集聚现象的研究

用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、阴极聚丙烯酰胺凝胶电泳和高效凝胶排阻色谱法, 研究了非还原脲变性蛋白溶菌酶在稀释复性过程中的集聚现象. 实验发现, 在整个稀释复性过程中, 没有蛋白溶菌酶集聚体沉淀产生. 当最终复性液中蛋白溶菌酶浓度小于4.0 mg/mL时, 复性过程中不会形成蛋白溶菌酶分子集聚体; 当最终复性液中蛋白溶菌酶浓度介于4.0～8.0 mg/mL时, 复性过程中会形成由非共价相互作用所引起的蛋白溶菌酶二分子和三分子集聚体; 而当最终复性液中蛋白溶菌酶浓度大于8.0 mg/mL时, 复性过程中除了会形成二分子和三分子蛋白溶菌酶集聚体外, 还会形成四分子蛋白溶菌酶集聚体. 在此基础上, 结合文献, 对非还原脲变性蛋白溶菌酶的稀释复性过程进行了描述.     

脲或盐酸胍溶液中变性溶菌酶复性过程中集聚体的研究

建立在蛋白质变性-复性三态模型的基础上, 给出了一个描述在变性液中变性蛋白质复性时蛋白质浓度和其复性率的关系式. 通过这个关系式, 可以获得两个重要的描述蛋白质变性-复性体系特征的参数, 一个是包含在一个集聚体分子中的变性蛋白质的分子数目n, 另一个是蛋白质从原始态到形成集聚体过程中的表观集聚平衡常数K. 以三种溶菌酶在脲和盐酸胍溶液中的变性-复性过程对此方程进行了验证, 结果表明所给出的方程能够很好地描述三种溶菌酶在这两种变性液中的复性结果, 三种溶菌酶在两种变性液中有形成二分子集聚体的趋势. 变性溶菌酶在复性过程中的电泳和高效凝胶排阻色谱也同时能够监测到复性过程中集聚体的形成, 并且监测结果与上述方程所得的结果一致.     

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。     

用蛋白电泳和高效凝胶排阻色谱法分析还原脲变性蛋白溶菌酶稀释复性过程的集聚体

本文利用蛋白电泳和高效凝胶排阻层析法分析了还原脲变性蛋白溶菌酶稀释复性过程中的集聚体。当用复性液稀释复性还原脲变性蛋白溶菌酶时,会迅速产生可观量的沉淀。沉淀和上清液的不连续十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻层析分析结果表明,还原脲变性蛋白溶菌酶在稀释复性过程中除了能够复性成天然态蛋白溶菌酶分子外,还会形成可溶的蛋白溶菌酶分子二聚体和三聚体,二聚体和三聚体主要是靠分子间二硫键的错配连接而成的;可溶的蛋白溶菌酶分子二聚体之间通过非共价键相互作用而形成集聚体沉淀,而可溶的三聚体溶菌酶分子则仍处于复性液上清液中。     

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。     

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。     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

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.     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

高效疏水作用色谱法对还原变性溶菌酶的折叠研究

首次用高效疏水相互作用色谱(HPHIC)研究了还原变性溶菌酶(Lys)的复性。对还原变性Lys在3种疏水性不同的色谱柱上的复性情况进行了考察,发现还原变性Lys在疏水性最弱的XDM-GM1型色谱柱上的复性效率最高,当Lys质量浓度为2.0 g/L时,其复性效率可达到94.6%。     

溶液中变性剂浓度对蛋白溶菌酶复性的影响

Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n（m1 -m2） and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography.     

Studies on Aggregation Interaction between Reduced Denatured Egg White Lysozymes during Refolding Procedure in Urea Solution

The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.     

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。     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to a-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

Theinvestigationofproteinrenaturation,orrefoldinghasbecomeaveryhotpointinbothliquidchromatography(LC)andlifesciencefields.Besidesusualdilutionanddialysismethods,manynewmethodsconcernproteinrenaturationhavebeenpresented,suchasreversemicelles1,chaperones2,polyethyleneglycol(PEG)3,surfactant4andantibodies5etc.Oneoftheauthorssuggestedhighperformancehydrophobicinter-actionchromatography(HPHIC)tobeanewtoolforproteinrenaturation6.Areviewonproteinrenaturationwithliquidchromatography(LC)wasrecentl…