A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues

A new, simple and rapid procedure for the purification of high amounts of mitochondrial porins from different tissues of mammalia is described. The method consists in a single step hydroxyapatite/celite chromatography of Triton X-100 solubilized mitochondrial membranes. For optimal purification several factors are critical such as the absence of salts, a low protein/detergent ratio and an exact hydroxyapatite/celite ratio of 2:1.     

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE

Abstract Within gram-negative bacteria such as Escherichia coli , the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.     

A rapid method for the isolation of purified amyloplasts from wheat endosperm

A rapid method for the isolation and purification of amyloplasts from the endosperm of developing grains of Triticum aestivum L. has been developed. Cell-free amyloplasts were mechanically isolated from plasmolysed tissue, and then purified by low-speed centrifugation through a single layer of Nycodenz sedimenting onto a cushion of agar. Recovery of amyloplasts was greater than 20% with less than 1% contamination by cytosol, 0.2% by mitochondria, 0.5% by endomembrane system and no contamination by microbodies. This method yields preparations which are routinely 55–65% intact up to 2 h after extraction. Amyloplast integrity was shown to depend upon the external sorbitol concentration, and amyloplastic enzymes in intact preparations were protected from digestion by trypsin.Abbreviations APPase alkaline pyrophosphatase - BSA bovine serum albumin - Hepes 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - (PPi)PFK pyrophosphate; fructose-6-phosphate 1-phosphotransferase Financial support for this research was provided by the Science and Engineering Research Council. The authors gratefully acknowledge many helpful discussions and initial assistance for this work from Professor T. ap Rees, Botany School, University of Cambridge, UK.     

A protocol for high throughput methods for the expression and purification of inner membrane proteins

The production of diffraction quality crystals for the structural determination of inner membrane proteins relies on obtaining large amounts of stable protein. Achieving this, by finding the correct parameters to successfully express and purify these proteins is often time-consuming and frustrating. The methods described here examine the most important parameters, in both expression and purification, quickly and simply. They take into account methods previously used in successful structural determinations of inner membrane proteins and collect and analyse data for use in further experiments and to investigate overall trends. These methods make use of histidine-tagged membrane proteins with a green fluorescent protein fusion but could be adapted easily for other proteins.     

一种提纯粗茶皂素的简易方法

对粗茶皂素的提取纯化工艺进行了研究。以质量分数为70％的粗茶皂素为原料,经2％NaOH溶解、盐酸酸析、95％乙醇溶解、丙酮沉析工艺得到精制茶皂素。检测结果表明,茶皂素质量分数大于95％,提取率大于80％,这种简便易行的工艺得到的茶皂素可作为开发新型植物灭螺剂的原料。     

小量快速浓缩病毒蛋白的研究

采用甘油透析浓缩及ＰＥＧ－６０００沉淀相结合的方法,对细胞病毒培养液进行处理,获得了较为纯化的病毒蛋白。该方法适用于病毒蛋白小量快速分析。纯化的病毒蛋白回收率约为５３μｇ／ｍｌ。     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.     

A simple method for the purification of over-expressed extracellular sucrase of Zymomonas mobilis from a recombinant Escherichia coli

The over-expressed extracellular sucrase (SacC) of Zymomonas mobilisfrom a recombinant Escherichia coli (pZSP62) carrying the sacC gene was purified partially by repeated cycles of freezing and thawing. This method separated the highly expressed recombinant protein from the bulk of endogenous E. coli proteins. The enzyme was further purified 14 fold with a 55% yield from the cellular extract of E. coli by hydroxyapatite chromatography. The purified enzyme had a Mr of 46 kDa by SDS-PAGE. Its km value for sucrose was 86 mM and was optimal at pH 5.0 and at 36°C.     

A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli

A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.     

A simple method for the purification of mitochondrial DNA from Plasmodium falciparum.

Mitochondrial DNA (mtDNA) from Plasmodium falciparum was isolated by conventional differential centrifugation in an SS34 rotor, a simpler method than CsC1 centrifugation of total DNA as employed by other workers. The nature of the sample was verified by sequencing a polymerase chain reaction (PCR) product obtained using oligonucleotide primers derived from known malarial mtDNA sequence.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.     

A simple method for large-scale purification of nucleic acids from plants using calcium phosphate-type monetite

Curently, the literature describes several nucleic acids purification methods, depending on the application and the required level of purity. These methods range from simple to complex and are mostly adapted for relatively small scale preparations. As an alternative, we developed in the present work an efficient, rapid, and up-scalable nucleic acids purification method based on the synthesis of a solid Calcium Phosphate-Type Monetite support (CPTM). The synthesis of the CPTM was optimized with regards to the calcium/phosphate (Ca/P) ratio and to sonication parameters (amplitude and time), and phase purity was resolved using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Analysis revealed the crystalline purity of the monetite phase and the identity of the matrix, and showing no secondary phases. Nucleic acids adsorption to the CPTM matrix was assessed under optimal conditions of buffering, ionic strength, pH, and flow rate, and the elution was carried out through a phosphate ions gradient that allowed an earlier elution of contaminants. We applied this purification method on several plants material, and results demonstrate that CPTM is a good matrix for nucleic acids purification from complex biological and environmental samples     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway? recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio? fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

A simple method for efficient separation and purification of c-phycocyanin and allophycocyanin from Spirulina platensis

c-Phycocyanin and allophycocyanin were separated and purified from Spirulina platensis by precipitation with ammonium sulphate, ion exchange chromatography and gel filtration chromatography. Pure c-phycocyanin and allophycocyanin were finally obtained with an A₆₂₀/A₂₈₀value of 5.06 and an A₆₅₅/A₂₈₀ value of 5.34, respectively.     

A rapid method for measuring organelle-specific substrate transport in homogenates from plant tissues

A rapid method is described for measuring organelle-specific metabolite transport systems in crude homogenates from plants. The tissues were homogenized in liquid nitrogen, extracted with buffer and reconstituted into artificial membranes. The method allowed demonstration of the known different substrate specificities of chloroplast triose phosphate/phosphate translocators from C₃- and C₄-plants, of the triose phosphate/phosphate translocator from non-green tissue, and of the dicarboxylate translocator. It thus by-passes the necessity to isolate intact plant organelles and, in addition, only a low amount of tissue material is required for transport measurements.Abbreviations Chl chlorophyll - TPT triose phosphate/phosphate translocator Dedicated to Professor F.-C. Czygan on the occasion of his 60th birthdayThis research was supported by the Bundesministerium für Forschung und Technologie. A.W. is a recipient of a Ph.D. fellowship from the Deutsche Forschungsgemeinschaft.     

A simple and reliable method for rapid production and purification of Pseudomonas aeruginosa haemolytic phospholipase C

AIMS: To design a simple method to produce active recombinant Pseudomonas aeruginosa haemolytic phospholipase C (PLC). METHOD AND RESULTS: Pseudomonas aeruginosa PLC is a virulence factor mainly involved in inflammatory and cytotoxic responses. While ammonium sulphate purification requires large amounts of bacterial suspensions and leads to low yields, production of recombinant protein in Escherichia coli is no more successful because of frequent inclusion bodies and accumulation of inactive PLC in the periplasmic space. Using an inducible system based on the glucose-repressed inv1 promoter in the yeast Schizosaccharomyces pombe, we were able to produce up to 10 IU ml(-1) of pure toxin within 24 h. CONCLUSIONS: This work describes the first method to easily get recombinant haemolytic PLC. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a powerful tool to study the mechanisms leading to its cellular toxicity.     

A simple method for purification of chymase from rat tongue and rat peritoneal cells

Chymase was purified from rat tongue and rat peritoneal cells by a simple new method involving hydrophobic chromatography on octyl-Sepharose 4B and hydroxylapatite column chromatography. This procedure can be completed in 1 or 2 days and the recovery is 45-60% from rat tongue and 32-47% from rat peritoneal cells. The specific activity of the purified enzyme is higher than that of crystallized enzyme previously reported (Y. Sanada, N. Yasogawa, and N. Katunuma (1978) Biochem. Biophys. Res. Commun. 82, 108-113). This procedure should be particularly useful for purifying chymase on a large scale from tissues in which it is present in relatively low concentrations.