			Home About us Contact

Protein Purification (protein + purification)

Distribution by Scientific Domains

Chemistry	56%
Life Sciences	26%

Distribution within Chemistry

Crystallography	23%

Selected Abstracts

Small-Molecule Affinity Ligands for Protein Purification: Combined Computational Enrichment and Automated In-line Screening of an Optically Encoded Library,

ANGEWANDTE CHEMIE, Issue 20 2010
Jakob
Hand in Hand: Eine vielseitige Methode zur Entwicklung niedermolekularer Affinitätsliganden wird beschrieben. Die Kombination aus Datenbankanreicherung mit automatisiertem Screening und Affinitätskartierung einer optisch codierten kombinatorischen Bibliothek ermöglichte die Identifizierung eines neuen Satzes von Liganden für die einstufige Aufreinigung des menschlichen Wachstumshormons (siehe Bild). [source]

Design of Affinity Tags for One-Step Protein Purification from Immobilized Zinc Columns

BIOTECHNOLOGY PROGRESS, Issue 1 2000
Richard S. Pasquinelli
Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichiacoli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions. [source]

Protein Engineering Strategies for Selective Protein Purification

CHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2005
M. Hedhammar
Abstract When producing and purifying recombinant proteins it is of importance to minimize the number of unit operations during the purification procedure. This is accomplished by increasing the selectivity in each step. Due to the high selectivity of affinity chromatography it has a widespread use in protein purification. However, most target proteins lack a suitable affinity ligand usable for capture on a solid matrix. A way to circumvent this obstacle is to genetically fuse the gene encoding the target protein with a gene encoding a purification tag. When the chimeric protein is expressed, the tag allows for specific capture of the fusion protein. In industrial-scale production, extension of the target protein often is unwanted since it might interfere with the function of the target protein. Hence, a purification scheme developed for the native protein is desired. In this review, different fusion strategies used for protein purification are discussed. Also, the development of ligands for selective affinity purification of native target proteins is surveyed. [source]

Protein purification using chromatography: selection of type, modelling and optimization of operating conditions

JOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009
J. A. Asenjo
Abstract To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant , -1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (, -lactoalbumin, ovalbumin and , -lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE

ELECTROPHORESIS, Issue 17 2009
Few Ne Chew
Abstract Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%. [source]

PURIFICATION OF AMYLASE FROM TILAPIA BY MAGNETIC PARTICLE

JOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 1 2010
MING CHANG WU
ABSTRACT Recent development in magnetic carrier technology involves the use of nonmagnetic substrates attached to superparamagnetic particles forming functionally modified magnetic support to isolate a particular enzyme in a controllable magnetic field. In this study, the superparamagnetic particles were modified by epichlorohydrin and other agents to cross-link with starch to form the purification support. This affinity support was able to bind the amylase and was used in the purification of amylase from Taiwan tilapia. After ammonium sulfate precipitation of amylase from Taiwan tilapia, the modified superparamagnetic particles were able to purify the crude amylase by 20.78-fold with recovery of activity of 75.6%. The molecular weight of the amylase was estimated to be 66.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both crude and purified amylase reached an optimum at a pH of 8.0 and temperature of 50C, and the enzyme was stable between 20 and 50C. PRACTICAL APPLICATIONS Because of the rapid development of high technology such as carrier supports for enzyme purification, the development, research and application of magnetic carriers are timely needed. The present study demonstrated that the affinity superparamagnetic particles could be used as a carrier support to absorb and purify the amylase and that technology of affinity purification can be widely used in protein purification. Compared with the traditional chromatography used in the purification of proteins, this novel affinity superparamagnetic particle technology is rapid, has low operation cost, requires simple facilities, and involves easy separation and recovery of the enzymes. [source]

Oligohis-tags: mechanisms of binding to Ni2+ -NTA surfaces

JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2009
Steven Knecht
Abstract Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C - or N -terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni2+ -NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni2+ -NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (KD) of 14,±,1,nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Nickel-Ion-Mediated Control of the Stoichiometry of His-Tagged Protein/Nanoparticle Interactions

MACROMOLECULAR BIOSCIENCE, Issue 2 2009
Mrinmoy De
Abstract The interaction between synthetic materials and biomolecules plays an important role in biomedical and pathological sciences. An important issue in these interactions is control of stoichiometry. The interaction between NTA ligands and proteins with six consecutive His residues has been widely used for protein purification. Control of stoichiometry is an important issue in applying this recognition strategy to the creation of defined nanoparticle-protein conjugates. In this communication we report the direct control of particle-protein stoichiometry through variation of nickel chloride concentration, as demonstrated through fluorescence and gel electrophoresis. [source]

Novel affinity tag system using structurally defined antibody-tag interaction: Application to single-step protein purification

PROTEIN SCIENCE, Issue 12 2008
Terukazu Nogi
Abstract Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody,peptide interaction, opening opportunities for further improvements/modifications. [source]

Identification of repertoires of surface antigens on leukemias using an antibody microarray

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2003
Larissa Belov
Abstract We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria. [source]

Popitam: Towards new heuristic strategies to improve protein identification from tandem mass spectrometry data

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2003
Patricia Hernandez
Abstract In recent years, proteomics research has gained importance due to increasingly powerful techniques in protein purification, mass spectrometry and identification, and due to the development of extensive protein and DNA databases from various organisms. Nevertheless, current identification methods from spectrometric data have difficulties in handling modifications or mutations in the source peptide. Moreover, they have low performance when run on large databases (such as genomic databases), or with low quality data, for example due to bad calibration or low fragmentation of the source peptide. We present a new algorithm dedicated to automated protein identification from tandem mass spectrometry (MS/MS) data by searching a peptide sequence database. Our identification approach shows promising properties for solving the specific difficulties enumerated above. It consists of matching theoretical peptide sequences issued from a database with a structured representation of the source MS/MS spectrum. The representation is similar to the spectrum graphs commonly used by de novo sequencing software. The identification process involves the parsing of the graph in order to emphazise relevant sections for each theoretical sequence, and leads to a list of peptides ranked by a correlation score. The parsing of the graph, which can be a highly combinatorial task, is performed by a bio-inspired algorithm called Ant Colony Optimization algorithm. [source]

Dwelling in the dark: procedures for the crystallography of phytochromes and other photochromic proteins

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009
Jo Mailliet
Crystallization of phytochromes and other photochromic proteins is hampered by the conformational changes that they undergo on exposure to light. As a canonical phytochrome, cyanobacterial Cph1 switches between two stable states upon absorption of red/far-red light. Consequently, it is mandatory to work in darkness from protein purification to crystal cryoprotection in order to ensure complete occupancy of one state or the other. With the simple and inexpensive methods that have been developed, phytochromes and other photochromic molecules can effectively be handled and crystallized, as has been demonstrated by the solution of the three-dimensional structure of the Cph1 sensory module. [source]

Structure of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with CDP- d -xylose

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2005
Nicole M. Koropatkin
Tyvelose is a unique 3,6-dideoxyhexose found in the O antigens of some pathogenic species of Yersinia and Salmonella. It is produced via a complex biochemical pathway that employs CDP- d -glucose as the starting ligand. CDP- d -glucose 4,6-dehydratase catalyzes the first irreversible step in the synthesis of this 3,6-dideoxysugar by converting CDP- d -glucose to CDP-4-keto-6-deoxyglucose via an NAD+ -dependent intramolecular oxidation,reduction reaction. Here, the cloning, protein purification and X-ray crystallographic analysis of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with the substrate analog CDP- d -xylose are described. Each subunit of the tetrameric enzyme folds into two domains. The N-terminal region contains a Rossmann fold and provides the platform for NAD(H) binding. The C-terminal motif is primarily composed of ,-helices and houses the binding pocket for the CDP portion of the CDP- d -xylose ligand. The xylose moiety extends into the active-site cleft that is located between the two domains. Key residues involved in anchoring the sugar group to the protein include Ser134, Tyr159, Asn197 and Arg208. Strikingly, Ser134,O, and Tyr159,O, sit within 2.9,Å of the 4,-hydroxyl group of xylose. Additionally, the side chains of Asp135 and Lys136 are located at 3.5 and 3.2,Å, respectively, from C-5 of xylose. In the structurally related dTDP- d -glucose 4,6-dehydratase, the Asp/Lys pair is replaced with an Asp/Glu couple. On the basis of this investigation, it can be speculated that Tyr159 serves as the catalytic base to abstract the 4,-hydroxyl proton from the sugar and that Asp135 and Lys136 play critical roles in the subsequent dehydration step that leads to the final product. [source]

Purification and characterization of Taq polymerase: A 9-week biochemistry laboratory project for undergraduate students

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010
Robert M. Bellin
Abstract We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of Thermus aquaticus DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week's procedure focus on a single learning goal. The laboratory series has been taught for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters. Statistical analysis of the survey results demonstrate that the laboratory series is very effective in teaching students the theory and practice of protein purification and analysis while also demonstrating positive results in more broad areas of scientific skill and knowledge. Amongst the findings, the largest reported increases in knowledge were related to students' understanding of how patent law relates to laboratory science, a topic of great importance to modern researchers that is readily discussed in relation to Taq polymerase. Overall, this laboratory series proves to be a very effective component in the curricula of undergraduate biology and chemistry majors and may be an appropriate laboratory experience for undergraduates. [source]

Improving Thai students' understanding of concepts in protein purification by using Thai and English versions of a simulation program,

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2007
Somkiat Phornphisutthimas
Abstract To support student learning in biochemistry and related courses, a simulation program, the Protein Purification Program, offers an alternative multimedia-based tool. This program has now been translated to produce a Thai version. However, translation from the original into the Thai language is limited by the differences between the language characteristics of English and Thai. Therefore, use of the program with Thai students had a twofold purpose. It helped their understanding of the concepts of protein purification by allowing code switching between the languages, but it also improved their understanding of, and competence in scientific English, which is a vital skill for functioning as a modern biochemist. According to the results of the questionnaires, undergraduates using the Thai/English program scored significantly higher than those using only the English language program (p < 0.05). In addition, the interview data suggested that the Thai/English program had improved student understanding of the concepts of protein purification to a greater extent than a single language (English) program. Students' overall preference in terms of their learning using the Thai/English program was 4.15 on a 1,5 Likert scale. [source]

A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009
Mats Ökvist
Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10,kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52,kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM,MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM,MtDS complex diffracted to 1.6 and 2.1,Å resolution, respectively. [source]

His-tagged protein purification by metal-chelate affinity extraction with nickel-chelate reverse micelles

BIOTECHNOLOGY PROGRESS, Issue 4 2010
Xiao-Yan Dong
Abstract Di(2-ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X-45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R2. The Ni(II)-chelate reverse micelles were characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R2 was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni2+ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two-stage purification by the metal-chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using ,-agglutinin for production of oral vaccines ,

BIOTECHNOLOGY PROGRESS, Issue 2 2010
Jamie L. Wasilenko
Abstract Yeast is an ideal organism to express viral antigens because yeast glycosylate proteins more similarly to mammals than bacteria. Expression of proteins in yeast is relatively fast and inexpensive. In addition to the convenience of production, for purposes of vaccination, yeast has been shown to have natural adjuvant activity making the expressed proteins more immunogenic when administered along with yeast cell wall components. Development of genetic systems to display foreign proteins on the surface of yeast via fusion to glycosylphosphatidylinositol-anchored (GPI) proteins has further simplified the purification of recombinant proteins by not requiring harsh treatments for cellular lysis or protein purification. We have expressed the hemagglutinin protein from a highly pathogenic avian influenza (HPAI) virus [A/Egret/HK/757.2/02], subtype H5N1, on the surface of the yeast strain Pichia pastoris, as an anchored C-terminal fusion with the Saccharomyces cerevisiae GPI-anchored cell wall protein, ,-agglutinin. Surface expression of the hemagglutinin fusion protein was demonstrated by immunofluorescence microscopy. Functionally, the fusion protein retained hemagglutinin agglutinating activity, and oral vaccination with the yeast resulted in production of virus neutralizing antibodies. This study represents the first steps in the generation of a yeast-based vaccine for protection against highly pathogenic strains of avian influenza. Published 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

A metal-chelate affinity reverse micellar system for protein extraction

BIOTECHNOLOGY PROGRESS, Issue 1 2010
Xiao-Yan Dong
Abstract A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X-45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X-45 less than 0.6, thermodynamically stable reverse micelles of water content (W0) up to 30 are formed. Di(2-ethylhexyl) phosphoric acid (HDEHP; 1,2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine-rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper-chelate reverse micelles (Cu-RM) are characterized for their W0, hydrodynamic radius (Rh), and aggregation number (Nag). Similar with reverse micelles of bis-2-ethylhexyl sodium sulfosuccinate (AOT), Rh of the Cu-RM is also linearly related to W0. However, Nag is determined to be 30,90 at W0 of 5,30, only quarter to half of the AOT reverse micelles. Then, selective metal-chelate extraction of histidine-rich protein (myoglobin) by the Cu-RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine-tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

Design of Affinity Tags for One-Step Protein Purification from Immobilized Zinc Columns

On-Chip Fragment-Based Approach for Discovery of High-Affinity Bivalent Inhibitors

CHEMBIOCHEM, Issue 5 2009
Isao Miyazaki
Abstract Covalent bonds not required: We describe a novel approach in which the concepts of fragment-based ligand discovery are combined with chemical array techniques to yield bivalent inhibitors. A pair of fragments is mixed and covalently attached to a glass slide by photolinking immobilization. The method does not require the compounds to have specific functional groups, and tedious steps for protein purification are avoided. Thus, the on-chip fragment-based approach is relatively simple and efficient for obtaining high-affinity ligands. [source]