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Chromatography Step (chromatography + step)
Selected Abstracts"In-gel patch electrophoresis:" A,new method for environmental DNA purificationELECTROPHORESIS, Issue 16 2005Changhyun Roh Abstract Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple, and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and polymerase chain reaction (PCR) amplifications. [source] Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 5 2007Julie Hardouin Abstract Proteins from biological samples are often identified by mass spectrometry (MS) with the two following "bottom-up" approaches: peptide mass fingerprinting or peptide sequence tag. Nevertheless, these strategies are time-consuming (digestion, liquid chromatography step, desalting step), the N - (or C -) terminal information often lacks and post-translational modifications (PTMs) are hardly observed. The in-source decay (ISD) occurring in a matrix assisted laser desorption/ionization (MALDI) source appears an interesting analytical tool to obtain N -terminal sequence, to identify proteins and to characterize PTMs by a "top-down" strategy. The goal of this review deals with the usefulness of the ISD technique in MALDI source in proteomics fields. In the first part, the ISD principle is explained and in the second part, the use of ISD in proteomic studies is discussed for protein identification and sequence characterization. © 2007 Wiley Periodicals, Inc., Mass Spec Rev 26:672,682, 2007 [source] Hydrogen/deuterium exchange on protein solutions containing nucleic acids: utility of protamine sulfateRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2008Anton Poliakov Obtaining global hydrogen/deuterium (H/D) exchange data on proteins is an important first step in amide proton exchange experiments. Important information such as the mode of exchange, the cooperativity of folding/unfolding reactions, and the effects of ligand binding can be readily obtained in global exchange experiments. Many interesting biological systems are complexes containing both proteins and nucleic acids. The low pH conditions required to quench H/D exchange reactions result in the formation of stable protein/nucleic acid precipitates which interfere with the liquid chromatography step of the experiment and preclude obtaining mass spectrometric data. In this work we show that the precipitation of proteins and nucleic acids is electrostatic in nature and can be prevented by high ionic strength and by removing nucleic acids by protamine sulfate. Using protamine sulfate in quenching solution, we were able to obtain global H/D data with protein samples containing large amounts of DNA or RNA. Copyright © 2008 John Wiley & Sons, Ltd. [source] Mechanism of antibody reduction in cell culture production processesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Yung-Hsiang Kao Abstract We recently observed a significant disulfide reduction problem during the scale-up of a manufacturing process for a therapeutic antibody using a CHO expression system. Under certain conditions, extensive reduction of inter-chain disulfide bonds of an antibody produced by CHO cell culture may occur during the harvest operations and/or the protein A chromatography step, resulting in the observation of antibody fragments (light chain, heavy chain, and various combination of both) in the protein A pools. Although all conditions leading to disulfide reduction have not been completely identified, an excessive amount of mechanical cell lysis generated at the harvest step appears to be an important requirement for antibody reduction (Trexler-Schmidt et al., 2010). We have been able to determine the mechanism by which the antibody is reduced despite the fact that not all requirements for antibody reduction were identified. Here we present data strongly suggesting that the antibody reduction was caused by a thioredoxin system or other reducing enzymes with thioredoxin-like activity. The intracellular reducing enzymes and their substrates/cofactors apparently were released into the harvest cell culture fluid (HCCF) when cells were exposed to mechanical cell shear during harvest operations. Surprisingly, the reducing activity in the HCCF can last for a long period of time, causing the reduction of inter-chain disulfide bonds in an antibody. Our findings provide a basis for designing methods to prevent the antibody reduction during the manufacturing process. Biotechnol. Bioeng. 2010;107:622,632. © 2010 Wiley Periodicals, Inc. [source] The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assemblyBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009B.M. Hartmann Abstract One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling ,-sheet forming peptide P11 -2 in fusion to thioredoxin. Homogenate was heat treated (55°C, 15,min) and then incubated with tobacco etch virus protease (TEVp) to release P11 -2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250,mM NaCl to homogenate to prevent P11 -2 from partitioning to the precipitate. This process structure gave recombinant P11 -2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials. Biotechnol. Bioeng. 2009; 104: 973,985. © 2009 Wiley Periodicals, Inc. [source] Large scale demonstration of a process analytical technology application in bioprocessing: Use of on-line high performance liquid chromatography for making real time pooling decisions for process chromatographyBIOTECHNOLOGY PROGRESS, Issue 2 2010Anurag S. Rathore Abstract Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real-time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale-up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale-up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Expression of Secreted His-Tagged S -adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and ImmobilizationBIOTECHNOLOGY PROGRESS, Issue 1 2008Yunxing Luo S -Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S -adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomycescerevisiae was successfully produced at high level (,200 mg/L) by the recombinant methylotrophic yeast Pichiapastoris. The secreted His6 -tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 °C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl -Met and ATP. [source] Expression and Purification of ZNF191(243,368) in Three Expression SystemsCHINESE JOURNAL OF CHEMISTRY, Issue 11 2007Dong-Xin ZHAO Abstract ZNF191(243,368), a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain adequate amount of ZNF191(243,368) for the study of its property, structure and function, three different expression systems of inclusion-body, glutathione S-transferase (GST), and hexahistidine (6×His) were used and compared. Among these systems, the expression level of ZNF191(243,368) was increased in inclusion body system under a higher isopropylthio- , - D -galactoside (IPTG) concentration, but the non-target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His-tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins. [source] A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysisELECTROPHORESIS, Issue 23 2007Hu Zhou Abstract The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome. [source] Haptoglobin from psoriatic patients exhibits decreased activity in binding haemoglobin and inhibiting lecithin-cholesterol acyltransferase activityJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 4 2008L Cigliano Abstract Objective, The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of haptoglobin (Hpt). Background, Hpt is a plasma acute-phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free haemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT). Study design, Hpt was isolated from patients with psoriasis vulgaris, and its activity in haemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. Methods, Two affinity chromatography steps, the first using resin-coupled haemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by Enzyme-linked immunosorbent assay with stationary haemoglobin or ApoA-I, Hpt in solution and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. Results, Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by haemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both haemoglobin binding and LCAT inhibition. Conclusions, In psoriasis, Hpt displays some structure modification(s), which might be associated with the protein function in the disease. [source] | |||||||||||||