蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议     

厌氧菌代谢产物的气相色谱和离子色谱分析方法比较

我们使用气相色谱和离子色谱分析方法对120株(包括9株标准菌株)厌氧菌的PYG培养物进行分析和比较,发现两种方法的分析结果类似,操作上各有优缺点。当然,相对来说离子色谱分析法更适于临床检验室和基层单位使用,因为离子色谱法基线稳定的时间短,水溶物标本不需预处理,也不用载气,操作简便,易于推广。     

两种水溶性抗菌活性物质的分离提取

对水溶性、不解离的极性物质分离时,一般采用吸附层析和凝胶层析等途径。实验通过硅胶柱层析、葡聚糖凝胶柱层析以及硅胶ＧＦ２５４制备型薄板层析,从发酵液样品中分离出两种有抗菌活性的纯物质。薄层层析的展开剂为二氯甲烷－四氢呋喃－甲醇－水（２５：３０：２）,分离出的组分中Ｒｆ＝０．７和Ｒｆ＝０．８两种物质有抗菌活性。硅胶柱层析洗脱过程为梯度洗脱,先用１５０ｍｌ上述展开剂洗脱,再用二氯甲烷－甲醇（２０：８０）     

Chiral counter-current chromatography: A survey of its instrument,mechanism, procedure,and applications

The chiral separation by counter-current chromatography has made great progress in the past three decades. It has become increasingly popular in the field of chiral separation, and many applications have been introduced during the last years. This review mainly focuses on the current topics, applications, and trends in chiral separation by counter-current chromatography. It contains the development of modern counter-current chromatography apparatus, theory of counter-current chromatography, overview of applications of chiral counter-current chromatography enantioseparation, its current situation, and challenges. At last, some conclusions and perspectives also have been discussed in this review.     

Isolation and Structure Elucidation of Three New Insecticidal Cyclodepsipeptides,Destruxins C and D and Desmethyldestruxin B,Produced by Metarrhizium anisopliae

A multi-channel continuous-flow analyzer equipped with biosensing devices was developed for multi-component measurement and its use in automating routine analysis was evaluated.

Biosensing was achieved by the aid of an immobilized enzyme reactor installed in the channel, and the channel switching process for the sensing of a different compound was made by using a column-switching rotary valve. Another rotary valve was used for auto-sampling. Both of the two rotary valves were interfaced to a system controller and work conjugatively in a programmed manner. Signal subtraction between different channels was found to be more precise compared with the multi-channel flow-injection analysis method, which is of merit for an analysis utilizing enzyme relay reaction (as for sucrose analysis) or for background signal subtraction. Glucose, lactate, and sucrose content in real samples were measured automatically with high reproducibility, and the results agree well with the kit method.     

A high-throughput approach to developing and optimizing mixed-mode membrane chromatography for protein purification

Membrane chromatography has been established as a viable alternative to packed-bed column chromatography for the purification of therapeutic proteins. Purification via membrane chromatography offers key advantages, including higher productivity and reduced buffer usage. Unlike column chromatography purification, the utilization of high-throughput screening in order to reduce development times and material requirements has been a challenge for membrane chromatography. This research focused on the development of a new, high-throughput screening technique for use in screening membrane chromatography conditions for monoclonal antibody purification. The developed screen utilizes a 96-well plate format, thereby allowing for the screening of multiple different membrane conditions at once. For this study, four mixed-mode cation exchange membranes and one cation exchange membrane were evaluated on the plate. The screen is performed in a similar manner to that of a resin slurry plate screen, however, instead of a single loading step, the antibody feed was loaded in 50 mg/ml increments up to a maximum loading of 450 mg/ml. Performing a similar, incremental loading on a resin plate would be impractical, as mixing times are substantially longer due to pore diffusion limitations. However, due to the significantly faster rate of mass transfer for membranes relative to resin, mixing times could be reduced by up to a factor of sixty on the membrane plate. Additional optimization showed that higher hydrophobicity can potentially lead to slower kinetics and mixing times that may need to be adjusted accordingly. The end result is a screen that has been proven to provide results comparable to those obtained on larger-scale membrane purification runs while also enabling exploration of a much greater operating space and significantly reducing the feed materials required.     

虎血清免疫球蛋白（IgG）的纯化及纯度、活性鉴定

目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。     

The influence of grafted polymer architecture and fluid hydrodynamics on protein separation by entropic interaction chromatography

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 +/- 0.005 chains/nm(2)) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities.     

重组人成骨蛋白-1的离子交换及分子排阻色谱法纯化及其复性研究

经发酵大量表达重组人成骨蛋白-1(rhOP-1)。SDS-PAGE发现rhOP-1表达量占细菌总蛋白的35%。菌体经裂解、洗涤后,用8mol/L尿素溶解包涵体,离心后提取目的蛋白。经离子交换色谱法对变性状态下的目的蛋白进行纯化,绝大部分杂蛋白被去除,目的蛋白纯度达93%以上。为进一步提高目的蛋白浓度,采用分子排阻色谱法对目的蛋白进行再次纯化,纯度达98%以上。利用降低尿素梯度的方法对纯化的蛋白进行复性,二聚体的含量在50%以上。Westernblot证明了复性后的目的蛋白以单体和有活性的二聚体的形式存在。     

Affinity-purification coupled to mass spectrometry: basic principles and strategies

Identifying the interactions established by a protein of interest can be a critical step in understanding its function. This is especially true when an unknown protein of interest is demonstrated to physically interact with proteins of known function. While many techniques have been developed to characterize protein-protein interactions, one strategy that has gained considerable momentum over the past decade for identification and quantification of protein-protein interactions, is affinity-purification followed by mass spectrometry (AP-MS). Here, we briefly review the basic principles used in affinity-purification coupled to mass spectrometry, with an emphasis on tools (both biochemical and computational), which enable the discovery and reporting of high quality protein-protein interactions.     

Analytical and preparative chromatographic procedures for obtaining pure cresyl violet and cresyl red from commercial cresyl violet

Abstract

Cresyl violet and cresyl red, components of commercial cresyl violet acetate, were separated and purified using preparative column liquid chromatography. The stationary phase was silica gel and gradient elution was carried out using chloroform:methanol. The purified dyes were obtained in high yield; 51% of the original lot was recovered as cresyl violet and 40% as cresyl red. Separated materials were characterized by nuclear magnetic resonance and mass spectroscopy; UV-visible and Fourier-transform infrared spectra also were obtained for samples of pure cresyl violet and cresyl red. The colored constituents of the commercial dye lot were identified using thin layer chromatography and reverse phase high performance liquid chromatography. Both methodologies were suitable for routine testing; reverse phase high performance liquid chromatography is an appropriate tool for quality control and high resolution identification of these compounds.     

Theoretical and experimental studies on zone-interference chromatography as a new method for determining macromolecular kinetic constants

Zone-interference chromatography is a new method for studying macromolecular interactions (S. Endo and A. Wada, Anal. Biochem. 124 (1982) 372). This method is a new style of affinity chromatography which requires no preparation of affinity-column materials but utilizes the velocity difference in a column between interacting molecular species. Using the stochastic theory on the behavior of solute molecules, both the association and the dissociation rate constants can be analytically obtained from the degree of deformation of elution patterns, i.e., the change of the first and second moments. In order to verify the present theory, computer simulation of elution profiles by the extended plate theory and a binding experiment between glutamate dehydrogenase and ADP have been carried out.     

Displacement to separate host‐cell proteins and aggregates in cation‐exchange chromatography of monoclonal antibodies

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host‐cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind‐and‐elute cation‐exchange chromatography (CEX) polishing step. A variant of the bind‐and‐elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%–1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low‐prevalence HCPs resulted in an effectively bimodal‐like distribution of HCPs along the length of a multi‐column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of low‐prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography.     

From Gel Filtration to Adsorptive Size Exclusion

Adsorption and size exclusion in starch and cross-linked dextran were phenomena discovered in Uppsala in the 1950s [Porath (1979), Biochem. Soc. Trans. 7, 1197; Porath (1981), Current Content 19, 21; Porath (1981), J. Chromatogr. 218, 241; Janson (1987), Chromatographia 23, 361; Laurent (1993), J. Chromatogr. 633, 1]. These discoveries were the background to the development of a variety of affinity chromatographic methods. At present attempts are being made to combine size exclusion chromatography (SEC) with adsorption into a single operation that we call adsorptive SEC (AdSEC).     

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.     

Affinity chromatography of neoglycoproteins

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.