从组合化学肽库中筛选亲和配基

在亲和色谱研究中 ,建立适合于某种分离对象的亲和色谱体系的关键是寻找适合的亲和配基 .小肽亲和配基具有性质稳定、合成简单、价格低及生物相容性好等特点[1] ,但这类配基也存在着亲和力弱或选择性低的缺点 .因此 ,如何寻找小肽配基以及如何提高小肽配基的亲和力和选择性的问题引起了人们的关注与重视 [1～ 3 ] .组合化学法是一种快速制备大量相关或同类化合物的革新性的方法 [4 ] ,组合肽库包括噬菌体展示肽库和合成肽库 ,这两类肽库均可用于小肽配基的筛选与优化 [1,5] .组合化学法尽管有效 ,但以小肽为目的物进行筛选时 ,往往因肽 -肽…     

高效亲和色谱研究含有特定结构域肽与DNA的结合

高效亲和色谱研究含有特定结构域肽与ＤＮＡ的结合张若蘅,杨栩,李崇熙,徐筱杰（北京大学化学系北京１００８７１）１前言在脱氧核糖核酸（ＤＮＡ）结合蛋白质中,除了螺旋－转折。螺旋、锌指和亮氨酸拉链三种主要结构域之外￣［１］,近年来,一类含有β－转折结构的Ｓ...     

磺胺甲基异恶唑作配基的高效亲和色谱分离胰凝乳蛋白酶和胰蛋白酶

用磺胺甲基异恶唑作基基，大孔硅胶和为基质的高效亲和色谱分离纯化胰凝蛋白酶及胰蛋白酶。２０多种蛋白质和酶在该柱上无特异性吸附。     

亲和色谱中配基的筛选与应用

亲和配基的选择与筛选是发展新的亲和色谱填料或构建一个新的亲和色谱体系所必须解决的首要问题。该文结合作者所在实验室的工作,对配基的选择、筛选与应用方面的一些进展进行了简要评述。作者所在实验室针对特定蛋白质和多肽的多肽亲和配基的筛选,开展了反义肽简并性的研究,发展了基于反义肽的组合化学筛选新方法。与常规的组合合成法相比,该方法简单、快捷、有效,极大地减小了合成和筛选的工作量,降低了筛选后亲和组分结构鉴定的难度。所建立的筛选策略已应用于流感病毒、严重急性呼吸道综合征(SARS)病毒亲和抑制剂的筛选和用于人β-干扰素测定的石英晶体微天平（QCM）生物传感器的构建,均取得了有意义的结果。     

基于天冬氨酸的固定化金属离子亲和色谱材料用于磷酸化肽选择性富集

采用点击化学的方法将自然界中的天冬氨酸(aspartic acid)键合到硅球上(命名为Click Asp),并将Fe3+配位到Click Asp上,合成固定金属离子亲和色谱(IMAC)材料(Fe3+-Click Asp);采用红外光谱、X射线电子能谱和扫描电镜等表征证明Fe3+-Click Asp成功合成。将此IMAC材料用于蛋白质酶解液和牛奶中的磷酸化肽的富集,实现了磷酸化肽的高选择性富集。本研究为磷酸化蛋白质组学提供了新材料和新方法。     

亲和色谱仿生配基的筛选和设计

利用选择性好、稳定性高、价格低廉的仿生配基作为亲和色谱的功能基团,实现对目的蛋白质的亲和分离,可以弥补常规的单克隆抗体和其他天然蛋白质配基在价格、稳定性方面的不足,从而拓展亲和色谱技术的应用范围。本文综述了亲和色谱仿生配基在筛选和设计方面的研究进展,重点介绍了组合方法和基于结构的仿生配基的设计及其应用。     

亲和色谱用无色合成配基对胰蛋白酶纯化功能的研究

考察了一种新型的阳离子无色三嗪化合物、几种阴离子无色三嗪化合物和三嗪染料作为亲和色谱配基对胰蛋白酶的吸附性能。猪胰蛋白酶经阳离子型配基一步纯化,比活达到１１６Ｕ／ｍｇ,纯化２１．６倍,回收率８８％。     

磺胺甲基异噁唑作配基的高效亲和色谱分离胰凝乳蛋白酶和胰蛋白酶

用磺胺甲基异噁唑作配基、大孔硅胶为基质的高效亲和色谱分离纯化胰凝乳蛋白酶及胰蛋白酶。20多种蛋白质和酶在该柱上无特异性吸附。磺胺甲基异噁唑在硅胶上的键合量为4．3mg／g；该亲和填料对胰凝乳蛋白酶吸附量为9．6mg／g；两者之间的亲和解离常数K1／M为1×103mol／L。     

新型镍离子亲和色谱介质的制备及其性能考察

以氯甲基化聚苯乙烯交联微球(氯球)为载体基质、N,N-二甲基甲酰胺(DMF)为溶剂、N,N-二异丙基乙胺(DIEA)催化,将合成的Lys(Boc)-OEt、氯乙酸乙酯连接到微球上。经水解使羧基裸露,与Ni²⁺螯合,制得新型镍离子亲和色谱介质。将其应用于His-Lys-Tyr三肽及Phe-His-Thr三肽纯化研究。以不含组氨酸的六肽作为杂质模型,用所制备的镍离子亲和介质进行分离。结果表明,所制备的镍离子亲和介质对两个含组氨酸的三肽具有很好的亲和作用,并实现了与不含组氨酸六肽的分离。     

高效液相色谱在生物医药研究中的应用（Ⅱ）肽化学研究中的高效液相色谱

七十年代中期以来.色谱学家逐步建立起包括反相HPLC的一整套色谱方法,并运用这些方法在肽的分离纯化,制备,定性定量分析,分子量测定,肽结构与其色谱保留值关系等方面进行了深入的研究。八十年代以来,HPLC在肽化学研究领域得到了广泛应用,取得突破性进展。主要表现在,人们利用HPLC并与其它方法相结合,发现了许多新的天然活性肽,特别是神经肽。如作者自83～88年不完全统计,已确定一级结构的新活性     

Electrochemical Evaluation of Binding Affinity for Aptamer Selection Using the Microarray Chip

Simultaneous evaluation of the binding affinity of a series of aptamers toward a target molecule was investigated using an electrochemical microarray chip. The chip was modified by immobilizing seven aptamers obtained from the SELEX process and a control sequence onto gold nanoparticle‐comprised conducting polymer‐coated microarray electrodes. The chip was then incubated with the target molecule, kanamycin. The electrochemical response of the captured kanamycin was studied to evaluate the binding affinity of the aptamers, which showed the same trend with the fluorescence spectroscopic results. The lowest dissociation constant between a selected aptamer and kanamycin was determined to be 38.06±0.73 nM.     

Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography

The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification problem. Different modes of chromatography that can cover almost every separation case are reviewed in this article. Additionally, an outlook to a very recent continuous chromatographic process (namely Multicolumn Countercurrent Solvent Gradient Purification, MCSGP) and future perspectives regarding purification strategies will be considered at the end of this review.     

Affinity Chromatography of Human Blood Coagulation Factor VIII on Monoliths with Peptides from a Combinatorial Library

FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.     

Design of Ligands for Affinity Purification of G-CSF Based on Peptide Ligands Derived from a Peptide Library

Introduction　　 Affinity chromatography is of great interest in pharmaceutical industry as it is simple,fast and is of a high efficiency to purify proteins from a complicated mixture to homogenousones in a single step. The most common affinity ligands are monoclonal antibodies,smallmolecules such as biotin and those specific to the bio-function of the protein of interest,suchas substrates and inhibitors. Although m Ab is less limited by the bioactivity of proteins,itspreparation is a complex …     

Tetraethyleneglycol diacrylate (TTEGDA)-crosslinked polystyrene resin as an efficient solid support for gel phase peptide synthesis

Various applications of the newly developed tetraethyleneglycol diacrylate (TTEGDA)-crosslinked polystyrene resin are illustrated by the synthesis of model peptides, fully protected peptides, peptide amides and biologically important sequences. PS-TTEGDA resin was prepared by suspension polymerization of styrene and TTEGDA and functionalized with chloromethyl, 4-cholromethyl-3-nitro, aminomethyl, α-bromopropionyl, a-aminopropionyl, 4-bromomethyl 3-nitrobenzamido, 4-aminomethyl-3-nitrobenzamido groups. Peptide synthesis was carried out using these modified resins by standard solid phase methodology. Coupling and deprotection in this synthetic strategy went to near completion showing the positive role of hydrophilic and flexible crosslinking agent TTEGDA in facilitating gelphase reactions. The peptides were removed from the support by photolysis, trifluoroaceticacid (TFA) treatment,trans-esterification or ammonolysis in high purity and yield. The crude peptides were purified by column chromatography/FPLC and characterized by aminoacid analysis, sequencing or¹H-NMR.     

液固色谱中溶质保留规律的研究—多元流动相组成对保留值的影响

根据热力学原理和简单的吸附色谱分子模型。本文提出了一个新的液固色谱保留模型，并导出了相应的保留值基本方程，以三元溶剂系统作为展开剂，用薄层色谱实验数据验证了所得方程的正确性。结果显示，计算Ｒｆ值与实验Ｒｆ值完全一致。     

藻类肝毒素的富集提取与分离

采用固相萃取（SPE）和高效液相色谱法（HPLC）对蓝绿藻肝毒素进行富集与分离。以悦目颤藻为研究对象,选择最佳方式破坏其细胞,使其释放出细胞内肝毒素,对肝毒素进行SPE富集提取。用反相HPLC技术,以C18柱作分离术,32%乙腈-水（含0.01mol/L乙酸铵）为流动相,达到了对藻类肝毒素的良好分离。