Intermediate filament systems are collapsed onto the nuclear surface after isolation of nuclei from tissue culture cells

Standard procedures for the biochemical isolation and purification of tissue culture cell nuclei are very similar to recently described methods for the preparation of detergent-resistant cytoskeletons. But the latter in addition to extracted nuclei contain cytoplasmic filament systems. We, therefore, investigated the distribution of these filamentous systems during nuclear isolation both by immunofluorescence microscopy and by SDS-gel electrophoresis. The intermediate filaments copurify with each single isolated nucleus. The majority of the filaments is collapsed onto isolated nuclei and still constitutes a filamentous system. This can be shown by partially unfolding the collapsed filament systems.     

木聚糖酶分离纯化技术

木聚糖酶应用广泛,其分离纯化是进行酶学性质研究、分子研究的前提条件,是成功确定氨基酸序列和三维结构的基础。综述微生物木聚糖酶分离纯化的方法,分析了常用方法在其分离纯化中的优缺点;强调了特异性分离纯化技术是高效的分离纯化方法;并对其它方法进行了概括。     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

Developments in techniques for the isolation,enrichment, main culture conditions and identification of spermatogonial stem cells

The in vitro culture system of spermatogonial stem cells (SSCs) provides a basis for studies on spermatogenesis, and also contributes to the development of new methods for the preservation of livestock and animal genetic modification. In vitro culture systems have mainly been established for mouse SSCs, but are lacking for farm animals. We reviewed and analyzed the current progress in SSC techniques such as isolation, purification, cultivation and identification. Based on the published studies, we concluded that two-step enzyme digestion and magnetic-activated cell sorting are fast becoming the main methods for isolation and enrichment of SSCs. With regard to the culture systems, serum and feeders were earlier thought to play an important role in the self-renewal and proliferation of SSCs, but serum- and feeder-free culture systems as a means of overcoming the limitations of SSC differentiation in long-term SSC culture are being explored. However, there is still a need to establish more efficient and ideal culture systems that can also be used for SSC culture in larger mammals. Although the lack of SSC-specific surface markers has seriously affected the efficiency of purification and identification, the transgenic study is helpful for our identification of SSCs. Therefore, future studies on SSC techniques should focus on improving serum- and feeder-free culture techniques, and discovering and identifying specific surface markers of SSCs, which will provide new ideas for the optimization of SSC culture systems for mice and promote related studies in farm animals.     

Separation of quinoxaline antibiotics by coil planet centrifugation

Liquid/liquid chromatography has been used for the isolation and purification of quinoxaline-type compounds on a coil planet centrifuge. Three solvent systems have been developed which are capable of high resolution both analytically and preparatively, Separations are described involving naturally occurring antibiotics, biosynthetically produced analogues, and chemically modified derivatives.     

Identification of Stimulants for Lactobacillus bulgaricus in Tomato Juice

Acid production in milk by Lactobacillus bulgaricus was stimulated by tomato juice or its serum. Preliminary purification of the stimulants involved adsorption and elution on a cation-exchange resin. Unidimensional paper chromatography in two solvent systems was employed in further isolation and purification. Identification of the stimulatory components was based on ultraviolet spectral analysis and thin-layer chromatography. The stimulants were identified as adenine and adenosine.     

Isolation and characterization of the proteolytic enzyme component from commercially available crude trypsins

A purification procedure for the isolation of a mixture of the major proteolytic pancreatic enzymes (trypsin, chymotrypsin, and elastase) from commercial crude trypsins is described. These enzymes are apparently the enzymes responsible for tissue dispersal in numerous cell culture systems. Materials toxic to cell cultures, present in certain crude trypsin samples, are removed during a purification involving centrifugation, dialysis, treatment with a cellulose ion-exchange resin, removal of salts, and lyophilization. While the fundamental use of this proteolytic mixture would be to prepare primary cell culture, the broad peptide bond specifleity of this mixture would suggest application in cases where a general protease, free of other enzymatic activities, is required.     

The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins

Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.     

Isolation and detection of protein with nano-particles and microchips for analyzing proteomes on a large scale basis

The advent of sugar-immobilized gold nano-particles (SGNPs), lipid-based nanoparticles, nano-chromatography and nano-electrophoresis has revolutionized the methodology for protein purification and proteomic research. This review provides an overview on the effective method developed for fast purification of protein from extracts using SGNPs. In addition, the current application of microfluidic systems for analytical purposes in biochemistry will also be explored that include the micro total analysis systems (μ-TAS) and lab-on-a-chip (LOC) analyses which are capable of isolation and detection of protein at the nanogram level. Finally, we describe why the lipid-based nano-particles (LBNPs) can enable the analysis in microchip electroseparation and how anionic and cationic LBNPs can be used for protein separation.     

Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics

Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.     

Purification techniques for human interferons

This article outlines the development and general status of present purification methods for human interferons. The isolation of each interferon species from natural sources is extremely difficult because of molecular characteristics, high losses of activity and the small amount of interferon protein present in production media. Few of the highly sophisticated methods meet the requirements for scale-up or give acceptable recoveries for a production process. The adaptation of purification procedures to the different interferon species is described, such as initial concentration, the extraction of beta interferon (IFN-β) by aqueous two-phase systems and the final purification of alpha interferon (IFN-α) and beta interferon to homogeneity. H.p.l.c. techniques are discussed in more detail together with problems in the purification of beta interferon and gamma interferon (IFN-γ). The range of interferon expression and excretion in recombinant microbial and animal cells is reviewed and different approaches for the solubilization and purification of intracellular recombinant interferons are described, which are covered mainly in patent applications.     

Foaming of proteins: New prospects for enzyme purification processes

Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the “White Biotechnologies” for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique.     

Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers

A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.     

The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia.

The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined. The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase. In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation. The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181). This strain was identified after a limited screening for lipase activity. About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.     

疫苗分离纯化研究进展

综述了国内外关于疫苗分离纯化的研究进展,系统介绍了目前可用作各类疫苗(尤其是基因工程疫苗)分离纯化的方法。沉淀和离心等传统分离技术在各类疫苗的分离纯化中应用广泛,层析技术和其它分离技术的结合已成为疫苗分离纯化的主流,新型膜技术和亲和层析在基因工程亚单位疫苗分离纯化中的作用引人注目。     

Characterization of an Inhibitor for Lactobacillus bulgaricus in Tomato Juice

Tomato juice (serum) added to milk in high concentration caused inhibition of acid production by Lactobacillus bulgaricus. The inhibitor was partially purified by adsorption on charcoal. Further isolation and purification involved paper chromatography in two different solvent systems. Ultraviolet-absorption spectra and thin-layer chromatography were used in characterization studies. The inhibitor was found to be a xylose- and adenine-containing nucleotide.     

Production of recombinant proteins in plant cells

Vegetable protein synthesis systems for industry, medicine, and research are becoming increasingly popular. The technology of protein production in plants has certain advantages, compared with the expression systems of bacteria and yeast. The rich variety of promoters, regulatory elements, affinity tags, and fusion partners that are used in molecular biology and plant biotechnology can create hybrid genetic constructs adapted to the solution of various tasks associated with protein synthesis and purification. New methods of modification of plant systems are being developed for the synthesis of functionally active human proteins whose structure is close to the natural analogues. This review shows current approaches to increase the yield of the target protein, facilitating the procedures of its isolation and purification and preventing degradation.     

A new look at xylanases: an overview of purification strategies

Interest in xylanases from different sources has increased markedly in the past decade, in part because of the application of these enzymes in the pulp and paper industry. Purity and purification costs are becoming important issues in modern biotechnology as the industry matures and competitive products reach the marketplace. Thus, new paths for successful and efficient xylanase recovery have to be followed. This article reviews the isolation and purification methods used for the recovery of microbial xylanases. Origins and applications of xylanases are described, highlighting the special features of this class of enzymes, such as the carbohydrate-binding domains (CBDs) and their importance in the development of affinity methodologies to increase and facilitate xylanase purification. Implications of recombinant DNA technology for the isolation and purification of xylanases are evaluated. Several purification procedures are analyzed, taking into consideration the sequence of the methods used in each and the number of times each method is used. New directions to improve xylanase separation and purification from fermentation media are described.     

实验动物胰岛细胞的分离纯化

胰岛移植作为治疗1型糖尿病的有效方法,临床应用前景较好。但是,由于在胰岛移植手术中,有功能的胰岛细胞数量较少,而严重影响其治疗效果。因此,如何提高胰岛分离纯化效果,获取尽可能多的高质量的胰岛,成为胰岛移植手术成败的关键。本文仅就在采用实验动物分离和纯化胰岛方面的实验经验作以简要介绍。     

From the neuroendocrinology of lymphocytes toward a molecular basis of the network theory

The cells of the immune system produce and respond to hormones that were once thought to be restricted to the neuroendocrine system. By applying a novel methodology based on the molecular recognition hypothesis, the isolation and purification of receptors shared between the immune and neuroendocrine systems was accomplished. Biochemical analysis revealed them to be virtually identical with respect to their physicochemical and functional properties. Thus, bidirectional communication between the immune and neuroendocrine systems seems to result from a common set of hormones and receptors which are shared by the two systems. Furthermore, the molecular recognition hypothesis has revealed that homologues of the binding sites of these same hormone receptor pairs may be contained within the hypervariable regions of immunoglobulins and therefore constitute part of the immunologic network.