二维液相色谱法在羊草地上部总蛋白分离中的应用

取8周龄羊草的地上部分,用三氯乙酸-丙酮法沉淀总蛋白,沉淀裂解后将缓冲液置换为起始缓冲液,进行第一维色谱聚焦分离。将第一维分离收集的pH值为8.5至4.0之间的组分分别进行第二维无孔硅胶反相高效液相色谱分离,利用ProteoVue软件获得羊草植株总蛋白pI/UV图谱,即羊草植株总蛋白质表达谱。文中对二维液相色谱法分离羊草蛋白质进行了方法学的研究,在第二维分离中尝试用3种不同的洗脱梯度条件进行分离,优化二维液相色谱分离条件并与传统凝胶双向电泳进行了比较,另外还对二维液相色谱的重现性和准确性进行了检验。实验建立了利用二维液相色谱分离羊草总蛋白的技术方法。     

大鼠附睾蛋白质组的提取和二维液相色谱分离

背景与目的：提取犬鼠附睾液蛋白并建立一种利用二维液相色谱法分离附睾蛋白组的方法。方法：分离提取犬鼠附睾液蛋白。样品利用起始缓冲液置换后,进行一维色谱聚焦分离,然后收集pH8．5—4．0之间的组份进行二维反相离压液相色谱分离,最后将获得的二维UV图通过ProteoVue软件转换成PI／UV图谱。结果：成功提取了附睾液蛋白,并通过二维液相色谱成功建立了大鼠头体尾部附睾液蛋白的二维PI／UV图谱,收集了一维色谱聚焦分离的pH8．5—4．0区间的20个组份,并将每个组份进行二维色谱分离后转换为PI/UV图谱。结论：为进一步全面研究附睾蛋白功能和体液差异蛋白质组研究打下了基础。     

多维液相色谱分离技术在复杂蛋白质组学样品鉴定中的应用

目的：随着蛋白组学技术的发展,液相色谱-串联质谱的联用技术（液质联用）逐渐成为蛋白组学的主流技术。方法：通过结合各种不同原理的色谱分离类型,多维液相色谱分离技术能够极大的提高分离系统的峰容量,达到有效分离复杂程度很高的蛋白质组学样品的目的。结果：最广泛使用的多维液相色谱分离系统是离子交换色谱（IEX）和反相色谱（RP）的二维结合,近年来又发展出了分离能力更强的三维液相色谱分离系统,并且已经在蛋白质组学研究中得到了应用。结论：本文综述了多种多维液相色谱分离方法,在这些方法中,不同的分离原理的色谱类型被用于肽段或蛋白混合物的预分离中,有效促进了样品的充分分离,极大地提高了复杂样品的蛋白组学鉴定能力。     

肠道病毒71型感染前后宿主细胞蛋白质组的二维液相色谱分离和比较

以肠道病毒71型及其宿主细胞为研究主体,建立了一种二维液相色谱分离和分析比较病毒感染前后细胞蛋白表达谱的方法。该方法以高效液相色谱(HPLC)为技术平台,对细胞裂解物先后进行一维色谱聚焦分离和二维反相色谱分离。利用ProteoVue软件将二维色谱数据转换成模拟胶图,再利用DeltaVue软件对感染前后的宿主蛋白表达谱进行比较和分析,找出差异蛋白。二维液相色谱分离法能够根据蛋白的等电点和疏水性建立精确的细胞蛋白表达图谱,每0．2个pH为一个收集区段,在pH8．5～3．9的范围内可见蛋白条带约1200条。该方法良好的重现性、自动化以及结果分析的简易化,使之在细胞表达谱差异显示中的应用潜力巨大,并且为研究病毒与宿主相互作用提供了新的方法和思路。     

绿豆非特异性脂转移蛋白结构与功能的关系分析

利用硫酸铵沉降、弱阳离子色谱、强阳离子交换色谱、高效液相离子交换色谱和凝胶色谱等方法从绿豆中分离纯化非特异性脂转移蛋白nsLTP,并培养出绿豆nsLTP的晶体。对绿豆非特异性脂转移蛋白结构与功能关系作了分析报道。     

AUT凝胶电泳在牛胰核糖核酸酶折叠研究中的应用

目的:牛胰核糖核酸酶是一种用于蛋白折叠研究的经典模式蛋白,在折叠研究过程中主要使用高效液相色谱用于分离检测不同阶段的蛋白折叠中间体。高效液相色谱具有自动化、分离效果好、样品可回收等优点,同时也存在检测通量较低、仪器设备较为昂贵等不足。AUT凝胶电泳简便、快捷、检测通量较高,本文尝试将其应用于牛胰核糖核酸酶的折叠研究。方法:使用AUT凝胶电泳、酶活性检测、质谱对牛胰核糖核酸酶还原变性过程及产生的折叠中间体进行检测;通过高效液相色谱和质谱对折叠中间单体进行分离检测,并分别进行AUT凝胶电泳检测以解析各折叠中间单体在电泳中的条带位置;通过AUT凝胶电泳和酶切后质谱鉴定各折叠中间单体的二硫键配对方式。结果:AUT凝胶电泳可以有效区分不同条件下的牛胰核糖核酸酶还原变性过程,检测结果与酶活性、质谱结果相符,并可以很好地区分牛胰核糖核酸酶还原变性过程折叠中间体。高效液相色谱将牛胰核糖核酸酶还原变性过程折叠中间体分离为13个色谱峰,并与AUT凝胶电泳中的11个条带位置进行匹配。确认牛胰核糖核酸酶还原变性过程折叠中间单体的二硫键配对方式,并与AUT凝胶电泳条带进行匹配,Cys58-Cys110和Cys26-Cys84构象熵减作用强于Cys40-Cys95和Cys65-Cys72。结论:AUT凝胶电泳适用于检测牛胰核糖核酸酶折叠中间体,可以与高效液相色谱、质谱等检测技术相互补充,共同应用于牛胰核糖核酸酶的折叠研究。     

二维液相色谱在中药分析的应用

中药的物质体系复杂,对其组分的分离、鉴定和制备需要更高的分离度,二维液相色谱能将分离机理不同而又相互独立的两支 色谱柱串联构成分离系统,应用于复杂基质的中药材及中药复方制剂的分析,可显著提高峰容量和色谱峰鉴定的可靠性,降低色谱峰重叠, 使分离效率与分析通量大为提高。综述二维液相色谱基本原理及其在中药分析中的应用研究新进展。     

制备液相色谱技术

液相色谱技术的发明,可以追溯到本世纪初。俄国植物学家茨维特以填装碳酸钙粉末的玻璃柱,分离植物色素的经典实验。自50年代之后,气相色谱、液相色谱等迅速发展,各种型式的色谱方法,成了分离和分析的利器,在科学技术的众多领域中发挥着重要作用。自70年代以来,高效液相色谱得到了飞速发展。     

制备型高效液相色谱法分离蛋白质的研究

生物工程的发展,特别是生化制品下游处理技术的兴起,对现代分离科学提出了更高的要求,研究和开发各类生化物质特别是活性生物大分子的分离纯化技术,已成为一项十分重要的研究课题。在现有的分离技术中,液相色谱,尤其是80年代在分析型高效液相色谱(HPLC)基础上兴起的制备型HPLC,在大规模分离纯化生物活性物质特别是蛋白质方面已显示出巨大的应用潜力,引起了各国研究者的高度重视^[1-5].本文利用自行设计的制备型HPLC分离装置,对牛血清白蛋白(BAS)和牛血清红蛋白(HG)的制备分离过程进行了实验研究,着重考虑了流动流速、柱超载方式、柱长等因素对BAS和HG分离度的影响。     

高效液相色谱技术在药品检验中的应用及进展

高效液相色谱法是20世纪中后期发展起来的一种新型的用于分离分析的色谱方法。具有分离速度快、分离效率高、灵敏度高及自动化程度高等优点,被广泛应用在生命科学领域和化学化工领域。近年来,高效液相色谱法在药品检验中也得到广泛应用,发挥了巨大的作用。本研究从高效液相色谱的简介、高效液相色谱法在药品检测中的应用等方面进行综述,目的是为我国药品检测提供新思路。     

Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer.

A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.     

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.     

Purification of membrane proteins in SDS and subsequent renaturation

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.     

The human platelet membrane proteome reveals several new potential membrane proteins

We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research.     

Protein binding to polymer brush,based on ion-exchange,hydrophobic, and affinity interactions

The major limitations associated with conventional packed bed chromatography for protein separation and purification can be overcome by using adsorptive microporous membranes as chromatographic media. Microporous membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate diffusion limitations. As a result, higher throughput and shorter processing times are possible using these membrane systems. In this paper, we review the current state of development in the area of attaching functionalized polymer brushes onto a microporous membrane to form a novel chromatographic medium for protein separation and purification. The functionalized polymer brushes were appended onto the pore surface of a microporous hollow-fiber membrane uniformly across the membrane thickness by radiation-induced graft polymerization and subsequent chemical modifications. We review various applications of this adsorptive membrane chromatography by focusing on polymer brushes bearing ion-exchange, hydrophobic and affinity groups. Proteins were captured in multilayers by the ion-exchange group-containing polymer brushes due to the formation of a three-dimensional space for protein binding via the electrostatic repulsion of the polymer brushes. In contrast, proteins were captured in a monolayer at most by the polymer brushes containing hydrophobic or affinity ligands. By permeating a protein solution through the pores rimmed by the polymer brushes, an ideal capturing rate of the proteins with a negligible diffusional mass-transfer resistance was achieved by the functionalized polymer brushes, based on ion-exchange, hydrophobic, and affinity interactions.     

Simultaneous ultrafiltration and affinity sorptive separation of proteins in a hollow fiber membrane module

A protein separation scheme combining affinity or ion exchange sorption with hollow fiber cross-flow filtration is described. Sorptive gel particles were loaded into the shell side of a hollow fiber membrane module. In the adsorption step, crude protein mixtures were passed through the lumen and permeating proteins passed through the membrane to bind on the gel particles in the shell. During elution, a buffer of adequate ionic strength to desorb the bound proteins was passed through the lumen and permeated through the shell. The eluant was then collected at the outlet to the shell of the hollow fiber module. The concept is illustrated by two examples: the purification of butyrylcholinesterase (EC 3.1.1.7) from raw horse serum using the affinity gel procainamide-Sepharose as the packing and the separation of carboxylesterase (EC 3.1.1.1) from beef liver homogenate using DEAE-Sephadex as the packing. The technique has the advantage of high volumetric throughputs typical of hollow fiber membrane modules as well as the high capacity characteristic of chromatographic packings. In addition, cross-flow filtration of particulates, agglomerates, and debris in passing protein from lumen to shell side can help eliminate the need for extensive pretreatment.     

Fast and efficient protein purification using membrane adsorber systems

The purification of proteins from complex cell culture samples is an essential step in proteomic research. Traditional chromatographic methods often require several steps resulting in time consuming and costly procedures. In contrast, protein purification via membrane adsorbers offers the advantage of fast and gentle but still effective isolation. In this work, we present a new method for purification of proteins from crude cell extracts via membrane adsorber based devices. This isolation procedure utilises the membranes favourable pore structure allowing high flow rates without causing high back pressure. Therefore, shear stress to fragile structures is avoided. In addition, mass transfer takes place through convection rather than diffusion, thus allowing very rapid separation processes. Based on this membrane adsorber technology the separation of two model proteins, human serum albumin (HSA) and immungluboline G (IgG) is shown. The isolation of human growth hormone (hGH) from chinese hamster ovary (CHO) cell culture supernatant was performed using a cation exchange membrane. The isolation of the enzyme penicillin acylase from the crude Escherichia coli supernatant was achieved using an anion exchange spin column within one step at a considerable purity. In summary, the membrane adsorber devices have proven to be suitable tools for the purification of proteins from different complex cell culture samples.     

Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process

Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis–sodium dodecyl sulfate (nrCE–SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used during the CE–SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE–SDS versus SDS–PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.     

Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.     

The expression of outer membrane proteins for crystallization

The production of sufficient amounts of chemically and conformationally homogenous protein is a major requirement for successful crystallization and structure determination. With membrane proteins, this constitutes a particular problem because the membrane volume is limited and the organisms are usually very sensitive to changes in membrane properties brought about by massive protein insertion. Moreover, the extraction of membrane proteins from the membrane with detergents is generally a harsh treatment, which gives rise to conformational aberrations. A number of successful procedures for functional expression followed by purification are reviewed here together with nonfunctional expression into inclusion bodies and subsequent (re)folding to produce functional proteins. Most of the data are for prokaryotic outer membrane proteins, but the outer membrane proteins of eukaryotic organelles are also considered as they do show similar features.