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

The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of a-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity recovery of a-Chy were found to be 25.1% for THFA, 10.4% for PEG, respectively. The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of a-Chy in high performance hydrophobic interaction chromatography (HPHIC) than that containing PEG, when the concentration of THFA was 3.2%, the bioactivity recovery of a-Chy is the highest.

Isoentropic and Isoenthalpic Temperatures of Protein Unfolding in Hydrophobic Interaction Chromatography

The thermal behaviors of five proteins in hydrophobic interaction chromatography （HIC） were investigated in the temperature range from 0 to 50℃. The thermodynamic parameters （△H°,△S°, △Cp°and △G°） of these proteins in the process of retention and unfolding were determined. The existence of enthalpy and entropy convergence with temperature was confirmed. The differences of the isoentropic and isoenthalpic temperatures for protein unfolding in HIC system from the traditional solution were elucidated.

Effect of ligand structure of stationary phase of high performance hydrophobic interaction chromatography on renaturation efficiency of GuHCl-denaturedα-chymotrypsin

The renaturation of the denatured α-chymotrypsin (α-Chy) with 1.7 mol · L-1 guanidine hydrochloride (GuHCI) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) with a comparable hydrophobicity but different ligand structures was investigated. The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency of α-Chy in the order of the end ligands PEG-600＜ phenyl group ＜ tetrahydrofurfuryl alcohol (THFA).

Applying flexible molecular docking to simulate protein retention behavior in hydrophobic interaction chromatography

Interaction between proteins and stationary phase in hydrophobic interaction chromatography (HIC) is differentiated into two thermodynamic processes involving direct nonbonding/conformation interac- tion and surface hydrophobic effect of proteins, hence quantitatively giving rise to a binary linear rela- tion between HIC retention time (RT) at concentrated salting liquid and ligand-protein binding free en- ergy. Then, possible binding manners for 27 proteins of known crystal structures with hydrophobic ligands are simulated and analyzed via ICM flexible molecular docking and genetic algorithm, with re- sults greatly consistent with experimental values. By investigation, it is confirmed local hydrophobic effects of proteins and nonbinding/conformation interaction between ligand and protein both notably influence HIC chromatogram retention behaviors, mainly focusing on exposed portions on the protein surface.

Retention model of protein for mixed-mode interaction mechanism in ion exchange and hydrophobic interaction chromatography

A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a 'U' shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.

Studies on the Renaturation with Simultaneous Purification of Recombinant Human Proinsulin with Unit of Simultaneous Renaturation and Purification of Protein in Semi-preparative Scale

The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (1050 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.

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 scales. That unit for the simultaneous renaturation and purification of proteins (USRPP) had the following four functions: to completely remove denaturant, to renature proteins, to separate renatured proteins from impurities, and to easily recycle waste denaturant. The efficiencies of refolding and purification of proteins by the USRPP are almost comparable to a usual long chromatographic column in laboratory. In preparative scale, USRPP can be easily, rapidly, and economically applied requiring a low pressure gradient. As an example, recombinant human interferon-? is employed to elucidate the application of the preparative USRPP.

A Novel Method for Diminishing Protein Aggregation during Denatuaration Process

The addition of packing material for high performance hydrophobic interaction chromatograghy （HPHIC） into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin （α-Chy） denatured with guanidine hydrochloride （GuHCl） solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.

一种疏水色谱填料的特性及应用的研究

以交联壳聚糖为基质 ,正戊醛为配基 ,利用改进的方法制备了疏水作用色谱 (HIC)填料 ,并对该色谱填料的吸附行为和应用作了研究。结果表明 ,此类填料对蛋白质的吸附行为符合疏水相互作用理论 ,对α 淀粉酶的纯化活性回收率大于 80 %。

金属螯合亲和色谱中的疏水作用

通过考察盐溶盐和盐析盐浓度对蛋白质在IDA裸柱和金属螯合柱上保留行为的影响,详细研究了金属螯合色谱中的疏水作用,疏水作用的发生、形成的条件以及不同条件下对蛋白质保留值的贡献。实验结果表明,在高浓度和低浓度的盐溶盐以及低浓度盐析盐中,蛋白质在金属螯合柱上的保留主要受静电和配位作用控制,而疏水作用对蛋白质的保留影响很小。对弱亲和性的金属螯合柱以静电作用为主,其大小可用参数Q表征;对强亲和性的IDA-Cu(Ⅱ)柱以配位作用为主。仅在高浓度的盐析盐中,金属螯合柱才呈现较强的疏水作用,支配蛋白质保留。实验证明,金属螯合色谱中疏水作用主要来自固定相间隔臂中的疏水碳链和盐析盐对蛋白质的增疏作用,利用这种疏水作用有可能改善金属螯合色谱分离的选择性。

用参数Z表征疏水色谱中脲浓度与蛋白质分子的构象变化

研究了５种标准蛋白在流动相中含有不同脲浓度条件下的疏水色谱保留行为。当脲 浓度不变时，蛋白质的保留仍然服从计量置换保留模型，并可测定在该特定脲浓度条件下蛋白质的Ｚ值。计量置换参数Ｚ可作为疏水色谱中生物大分子构象变化的表征。蛋白质的Ｚ值随脲浓度的增大而减小。除分子构象变化的因素外，Ｚ值变化与脲参与置换剂分子间的计量置换过程有关。Ｚ值变化的连续性与否直接反映了蛋白质分子构象的连续性变化或突变。

蛋白质在疏水色谱中的变性热力学

研究21～80℃温度范围内一些蛋白质和小分子在疏水相互作用色谱中的热行为.利用Van＇t Hoff作图(lnk＇-1/T)测定蛋白质分子的热力学参数(△H°,△S°和△G°),根据标准熵变(△S°)和标准自由能变(△G°)判断蛋白质在色谱过程中的构象变化,通过△H°-△S°的线性关系估计蛋白质变性时的“补偿温度”(β),鉴定蛋白质在疏水相互作用色谱中保留机理的同一性.

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。

用高效疏水色谱法对多种脲变α-淀粉酶折叠中间体的研究

用高效疏水色谱法对用脲变性的α-淀粉酶的体外折叠中间体进行了分离，发现脲变α-淀粉酶折叠至少有19个中间体，而且，这些中间体在色谱流出液中可稳定一周．这一结论已由电泳、离子交换色谱和体积排阻色谱法证实．此外，还用紫外吸收光谱和荧光发射光谱研究了这些折叠中间体与天然α-淀粉酶构象之间的差异．

重组人干扰素-γ的制备与鉴定

用聚乙二醇200疏水相互作用色谱固定相(PEG200-STHIC)分别在色谱柱和色谱饼上完成了一步复性并同时纯化来源于大肠杆菌(E.coli)表达的重组人干扰素-γ(rhIFN-γ)。为了能使色谱分离方法用于不同来源的rhIFN-γ的纯化,对rhIFN-γ在反相色谱、离子交换色谱、固定化镍离子亲和色谱上的保留行为也进行了研究。色谱柱纯化的rhIFN-γ收集液经排阻色谱除盐和冷冻干燥得到rhIFN-γ干粉。用基质辅助激光解吸电离飞行时间质谱对rhIFN-γ干粉进行了测定,rhIFN-γ单体的相对分子质量为17184.0,二聚体的相对分子质量为34204.4。用细胞病变抑制法(CPEI)测定rhIFN-γ干粉的比活性为9.5×108IU/mg。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定rhIFN-γ干粉的纯度高于95%。用色谱柱复性并同时纯化rhIFN-γ的质量回收率达到93.7%,纯度高于95%,比活性为4.3×107IU/mg。结果表明,采用PEG200-STHIC色谱柱复性并同时纯化rhIFN-γ是一种十分高效的方法。

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 。

使用疏水作用色谱研究蛋白质的构象变化

研究了高效疏水作用液相色谱中(HIC)色谱条件改变对蛋白质构象的影响。发现固定相配体的疏水性、温度及流动相中盐的阴离子、阳离子和pH值都影响蛋白质的构象。

疏水作用色谱填料研究进展

疏水作用色谱法（HIC）是一种有效的蛋白质分离纯化方法。综述了HIC填料的基质和配基的发展过程，举例说明了不同HIC填料对蛋白质的分离纯化效率，指出了制备型HIC填料的现状及其发展趋势。

疏水层析法复性重组人γ干扰素

重组人γ-干扰素(rhIFN-γ)在大肠杆菌中高效表达,并形成包涵体.利用疏水作用层析(HIC)法对rhIFN-γ进行复性.采用了等尿素梯度和线性尿素梯度两种复性方法,考察了线性尿素梯度下尿素梯度长度、尿素终浓度、流速和上样量对rhIFN-γ复性的影响.在优化的线性尿素梯度复性条件下,尿素浓度在10个柱体积内从6mol·L-1下降到2mol·L-1,流速为1ml·min-1、上样量为0.568mg时,rhIFN-γ的活性收率比稀释复性法提高6.5倍,蛋白质量收率为36%,比活达1.9×108IU·mg-1.

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAESephadexA50柱层析及PhenylSepharoseFastFlow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明:以0.25mol/LKSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。