New procedures for glycophorin A purification with high yield and high purity

Glycophorin A (GPA) is the major glycoprotein of the human erythrocyte membrane. It is known to form, in SDS gels as well as in a membrane environment, homodimers, and also heterodimers with the homologous molecule Glycophorin B (GPB). It is shown in this report that the propensity of GPA to dimerize with GPB precludes satisfactory preparation with high yield of pure GPA using classical techniques including SEC and RPLC. It was demonstrated using multiple angle light scattering that GPA is eluted from RPLC columns as dimers. A convenient procedure was devised which allowed us to get pure GPA with high yield. This procedure consists of selectively blocking GPA–GPB heterodimer formation by selective modification of Cysteine 50 of GPB before RPLC.     

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.     

New method to prepare single-headed heavy meromyosin with high purity and a high yield

We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.     

High-throughput laser-mediated in situ cell purification with high purity and yield.

BACKGROUND: Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield. METHODS: An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined high-throughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility. RESULTS: Photothermal purification required approximately 10(8) W/cm(2) at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 10(9) W/cm(2) at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 10(10) W/cm(2) independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed-loop process to re-image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5-100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B- and T-cell mixtures over a wide range of contaminating cell percentages (0.1-99%) and cell densities (10(4)-10(6)/cm(2)). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated. CONCLUSIONS: In situ laser-mediated purification was achieved with nonadherent and adherent cells on the automated laser-enabled analysis and processing platform. Closed-loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 10(1) to 10(8) cells. Throughput ranged from approximately 10(3) to 10(5) total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively.     

泛素、泛素链和蛋白质泛素化研究进展

蛋白质泛素化是以泛素单体和泛素链作为信号分子,共价修饰细胞内其他蛋白质的一种翻译后修饰形式。不同蛋白质底物、同一底物的不同氨基酸修饰位点以及同一位点上泛素链连接方式的不同均可导致细胞效应的差异。蛋白质泛素化在真核细胞内广泛存在,除了介导蛋白质的26S蛋白酶体降解途径之外,还广泛参与了基因转录、蛋白质翻译、信号传导、细胞周期控制以及生长发育等几乎所有的生命活动过程。泛素链的形成及其修饰过程的任何失调均可导致生物体内环境的紊乱,从而产生严重的疾病。文中结合实验室研究,综述了泛素的发现历史、基因特点、晶体结构,特别是泛素链的组装过程、结构、功能以及与人类相关疾病关系的新进展,可为这些疾病的治疗靶点和药物靶标的研究提供思路。     

Rapid purification of EGFP, EYFP, and ECFP with high yield and purity

Most current high throughput purification procedures for the green fluorescent protein (GFP) suffer from poor yields and low purity. An improved purification procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial fluorescent protein content) has been developed. The purification procedure requires only two steps: the cell lysate is heated to 60 degrees C for 4 min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same fluorescence profile as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan fluorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.     

Rapid separation of lipid classes in high yield and purity using bonded phase columns

A method utilizing aminopropyl bonded phase (Bond Elut) columns has been developed to separate lipid mixtures into individual classes in high yield and purity. Up to ten lipid mixtures can be processed in 1 hr and the columns are reusable after suitable washing. Although the method was developed with standard lipid mixtures, it was shown that it is also applicable to biological extracts. Due to the rapidity and high yields (greater than 95%) of this procedure, it is superior to preparative HPLC or TLC, or other chromatographic methods for the separation of lipid mixtures for subsequent analysis.     

Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.     

High purity, high yield procedure for butyrolactone I production from Aspergillus terreus

Two experimental design methods, Plackett-Burman and Box-Behnken, were used to optimize media for the production of butyrolactone I, a chemical inhibitor of eukaryotic cyclin-dependent kinases, synthesized by Aspergillus terreus. The optimized medium produced as much as ten fold more butyrolactone I than the original medium. An isolation procedure is also described which generates highly purified butyrolactone I, free from other secondary metabolites produced by this strain of A. terreus. The results of this study provide the means to produce highly purified preparations of butyrolactone I.     

A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil

A low expense process is developed for recovering esterified eicosapentaenoic acid (EPA) from microalgae and fish oil. Over 70% of the EPA content in the esterified crude extract of microalgae were recovered at purities exceeding 90%. The recovery scheme utilizes either wet or freeze-dried algal biomass. The process consists of only three main steps: 1) simultaneous extraction and transesterification of the algal biomass; 2) argentated silica gel column chromatography of the crude extract; and 3) removal of pigments by a second column chromatographic step. Argentated silica gel chromatography recovered about 70% of the EPA ester present in the crude fatty ester mixture of fish oil, but at a reduced purity ( approximately 83% pure) compared to the microalgal derived EPA. The optimal loading of the fatty ester mixture on the chromatographic support was about 3% (w/w) but loadings up to 4% did not affect the resolution significantly. The process was scaled up by a factor of nearly 320 by increasing the diameter of the chromatography columns. The elution velocity remained constant. Compared to the green alga Monodus subterraneus, the diatom Phaeodactylum tricornutum had important advantages as a potential commercial producer of EPA. For a microalgal EPA process to be competitive with fish oil derived EPA, P. tricornutum biomass (2.5% w/w EPA) needs to be obtained at less than $4/kg. If the EPA content in the alga are increased to 3.5%, the biomass may command a somewhat higher price. The quality of microalgal EPA compares favorably with that of the fish oil product. Compared to free fatty acid, EPA ester is more stable in storage. Shelf-life is extended by storing in hexane. The silver contamination in the final purified EPA was negligibly small (<210 ppb).     

Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

Procedure for isolation of gangliosides in high yield and purity: simultaneous isolation of neutral glycosphingolipids

While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.     

Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation

Several mechanisms have been proposed for the synthesis of substrate-linked ubiquitin chains. HECT ligases directly catalyse protein ubiquitination and have been found to non-covalently interact with ubiquitin. We report crystal structures of the Nedd4 HECT domain, alone and in complex with ubiquitin, which show a new binding mode involving two surfaces on ubiquitin and both subdomains of the HECT N-lobe. The structures suggest a model for HECT-to-substrate ubiquitin transfer, in which the growing chain on the substrate is kept close to the catalytic cysteine to promote processivity. Mutational analysis highlights differences between the processes of substrate polyubiquitination and self-ubiquitination.     

Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.     

Preparation of rat liver plasma membranes in a high yield

Existing procedures for the preparation of rat liver plasma membranes are time consuming and generally produce low yields. A method is described in which a rat liver homogenate low-speed pellet is fractionated on a self-forming Percoll gradient. Plasma membranes can be removed from the gradient in a high yield along with much of the DNA in the liver homogenate. A second Percoll step performed in the presence of a low concentration of calcium ions separates the DNA from the plasma membranes. The final membrane fraction has high specific activities of marker enzymes with little contamination with microsomal, mitochondrial, Golgi, or lysosomal markers.     

Preparation of polymer-supported benzyllithium reagents

Cross-linked polystyrenes (2, 8, and 11) possessing pendant double bonds conjugated with benzene ring were prepared by both polymerization method and chemical modification of pre-formed polymers. These double bonds were readily lithiated with n-butyllithium to give the benzyllithium species on the polymer. The polymer supported benzylic anions (3 and 12) are a versatile polymeric organometallic reagent that reacted with various kinds of electrophiles.