Protein purification and characterization pdf

Purification and characterization of a surface-binding protein from Lactobacillus fermentum RC14 that inhibits adhesion of Enterococcus faecalis Printed in Great Britain Purification and characterization of a. One of the major surface proteins of this fungus showed haemagglutinating activity and was isolated by affinity chromatography using a mucin Sepharose column.

Biochemical analysis showed that the protein was a dimeric glycoprotein with a molecular mass of 36 kDa and an isoelectric point of pH 6. The protein was N-terminally blocked; four internal peptides were sequenced, and showed no significant similarity to sequences in the Swiss-Prot or PIR databases.

The haemagglutinating activity of the isolated protein was not inhibited by any of the mono- or disaccharides tested, but it was inhibited by the glycoproteins fetuin and m u c h The haemagglutinating activity changed after incubating the protein in buffers of different pH, with maximal activity at pH The lectin was tested for different enzymic activities but none were detected.

Analysis of the haemagglutinating activity in various cell fractions indicated that the protein was associated with extracellular polymer layers and with the cell wall of the fungus. About the same amount of the haemagglutinating protein was recovered from samples of vegetative mycelium and of mycelium containing nematode-trapping cells.

Introduction a three-dimensional network Barron, ; Nordbring- Hertz, Flooding a colony of A. Small amounts of a GalNac- ment is still, however, limited Kennedy, , and no binding protein have been purified on a GalNac-affinity fungal adhesive compound has been fully characterized column Borrebaeck et al. Carbohydrate-protein Pramer, , and it has been found only in trap- lectin interactions have been proposed to mediate containing mycelium and not in vegetative mycelium adhesion in several fungal-host systems, plant patho- Borrebaeck et al.

Using a soybean gens, fungal-fungal interactions mycoparasitism , fun- agglutinin SBA ; specificity for GalNac affinity gel, gal-algal interactions and nematophagous fungi Borrebaeck et a!.

One of the first systems to suggest lectin-mediated Involvement of lectin-mediated adhesion was further adhesion was the nematophagus fungus Arthrobotrys confirmed by demonstrating that entrapment was re- oligospora. Recent studies of the adhesion mechanism in A. Roskn and others oligospora have indicated that the adhesion between Haemagglutinating activity in various cell fractions.

To obtain more traps and nematodes is a complex process involving both information about the localization of the lectin, the pellets remaining after the homogenization of the mycelium were fractionated further.

To further pooled. First, extracellular polymers were removed by sonication using understand the molecular background of the adhesion a slight modification of the method described by Tunlid et al.

The pellet was washed with PBS ing protein and reacts with antibodies prepared against 2 x 25 ml. The sonication was then repeated twice with 25 ml PBS the lectin previously isolated by GalNac affinity chroma- and intervening centrifugation.

The sonicated pellet was transferred to tography Borrebaeck et al. Acid-washed glass beads 0. The cell wall pellet was resuspended obtained when A. All the supernatants obtained during the purification mented with 0.

For production of hyphae without activity. Each culture vessel contained ml medium and was 1 a 5 ml Eppendorf tubes. The cell walls were agitated vortex mixer, inoculated with a water suspension of conidia prepared from a culture 90 s with 1 ml of an enzyme solution 1 mg ml-I ; PBS containing grown on corn meal agar Difco supplemented with 2 g K2HP04 1 - I.

Following incubation, the samples were centrifuged g, 10 min and the haemagglutinating activity in the Protein puriJication. All steps of the purification were carried out at supernatant was measured. Control samples were treated with PBS "C. Mycelium from two vessels 2 x ml was harvested after only.

The mycelium approx. Protein content. The protein content was determined using the mg dry wt was washed with 3 x 50 ml 20 mhl-sodium phosphate method of Bradford using bovine serum albumin as a standard. The Samples were diluted 1 : 1, by vol. The supernatant pH 6. Samples with or without Amicon, W. Grace, Helsingborg, Sweden. The relative showed no haemagglutinating activity. The mycelial pellets were molecular mass was calculated by using a molecular market kit LKB, frozen until used for the study of haemagglutinating activity in various Bromma, Sweden including ovotransferrin kDa , bovine serum cell fractions see below.

The gel was kept in Gelfiltration. Elution was followed at and nm background absorbance nm was detected. Protein bound to the and fractions were collected by a HeliFrac Pharmacia. The apparent column was eluted with freshly prepared 0. The protein was also isolated according to the procedure described by Borrebaeck et al. The tinating units. After preincubation for 1 h at room temperature, 20 p1 gel was stained with Coomassie Brilliant Blue. The column commercial preparations of monosaccharides can contain haemagglu- was equilibrated with 0.

The lectin was eluted with solutions were ultrafiltered through Centricon 10 membranes to Polybuffer 74, pH 5. The remove molecules larger than 10 kDa before being used in the chromatofocusing was performed at 21 "C with a flow rate of inhibition studies. Carbohydrate solutions that were not filtered were 1. O ml min-'. Protein bands values was determined as follows: 50 p1 protein dissolved in PBS reacting with rabbit antibodies prepared by C.

Borrebaeck against the pg ml-I was incubated with pl of either 0. This buffer was made by anti-rabbit antibodies Sigma , Blake et al. Purified lectin 25 pg was digested with Achromo- and adjusting the volume to ml with HzO. After incubating the bacrer lyticus protease Wako Pure Chemical Industries Ltd, Osaka, samples for 80 min at room temperature the buffers were changed to Japan in 2 M-guanidine hydrochloride essentially as described by PBS by ultrafiltration Centricon 10 and the haemagglutinating Riviere et al.

Hydroxylamine cleavage was carried out as activities were measured. The affinity-purified protein dissolved in PBS was 2. Pumps were System Gold Beckman and the detector assayed for various activities, using pg protein. Acid phosphatase was a photo diode-array Waters. Buffers used for the reversed EC 3. Protein Purification and Characterization Techniques. Uploaded by Jacqueline Rose Alipo-on. Document Information click to expand document information Description: biochem.

Did you find this document useful? Is this content inappropriate? Report this Document. Description: biochem. Flag for inappropriate content. For Later. Related titles. Carousel Previous Carousel Next. Characterization of Exopolysaccharides Produced by Jump to Page.

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Chapter 5 shows the purification of fission yeast Dmc1 and its accessory proteins, and describes a conventional method to monitor DNA strand exchange reaction, which is a powerful tool to understand the biological significance of Dmc1 as well as its accessory proteins. Chapter 7 summarizes mouse and human studies that provide mechanisms by which cholesterol could affect inflammation. Apart from the direct effects, its intracellular localization as well as the contribution of different types of cholesterol to the inflammatory response is highlighted -- when oxidized, cholesterol is more likely to instigate inflammation.

Chapter 8 summarizes major cell sources, important proteins, transcription factors and signaling cascades, which governs mesenchymal stromal cell MSC fate towards the osteogenic lineage as well as new trends in the development of scaffold materials with osteoconductive and osteoinductive properties. Chapter 9 describes features, purification methods and applications of proteins such as membrane bound proteins, enzymes or recombinant proteins produced by halophilic bacteria.

Chapter 10 discusses various tau modifications associated with tau aggregation. Tau aggregation is a pathological hallmark of many neurodegenerative diseases including AD. Chapter 11 discusses the properties of the Clostridium difficile toxins, the mechanism of action, and the immunopathogenesis of the toxins.

Clostridium difficile toxins will trigger Clostridium difficile infection CDI which is the leading cause of hospital-acquired and antibiotic-associated bacterial diarrhea in the United States. Chapter 12 discusses the design of bioseparation strategy for engineering purification of conjugated proteins. The strategy is built on physicochemical properties which include molecular size, surface charge distribution and relative hydrophobicity for size exclusion, ion exchange and hydrophobic interaction chromatography respectively.

Calcium plays an important role in a wide variety of biological processes. This divalent metal ion can bind to a large number of proteins; by doing so it modifies their biological activity or their stability. Because of its distinct che- cal properties calcium is uniquely suited to act as an on—off switch or as a light dimmer of biological activities.

The two books entitled Calcium-Binding Protein Protocols Volumes I and II focus on modern experimental analyses and methodologies for the study of calcium-binding proteins. Both extracel- lar and intracellular calcium-binding proteins are discussed in detail. H- ever, proteins involved in calcium handling e. Also, calcium-bi- ing proteins involved in bone deposition will not be discussed, as this specific topic has been addressed previously.

The focus of these two books is on studies of the calcium-binding proteins and their behavior in vitro and in vivo. The primary emphasis is on protein chemistry and biophysical methods.

Many of the methods described will also be applicable to proteins that do not bind calcium. Calcium-Binding Protein Protocols is divided into three main sections. The section entitled Introduction and Reviews provides information on the role of calcium in intracellular secondary messenger activation mechanisms. Mo- over, unique aspects of calcium chemistry and the utilization of calcium in dairy proteins, as well as calcium-binding proteins involved in blood clotting, are addressed.

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.

The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. The complexity and sheer number of proteins in a cell are impediments to identifying proteins of interest or purifying proteins for function and structure analysis. Thus, reducing the complexity of a protein sample or in some cases purifying a protein to homogeneity is necessary. There are totally three volumes. This book is the last volume.

Chapter 1 describes "in vivo" and "ex vivo" approaches for determining the role of an olfactory receptor protein in the detection of its cognate agonist and various analogs. Surprising responses of the olfactory receptor to unrelated compounds is also discussed.

Chapter 2 reviews the recent studies on the features of PTEN in the signalling pathways involved in several diseases as emerging evidences suggest that PTEN enzymatic activity will not cover the entire mechanism of the ability. Chapter 3 proposes site-directed mutagenesis approach for determining the structure-function relationships of neurotransmitter transporters. Therefore, bp N-terminal been considered as a promising vaccine candidate for nucleotides of exoA gene and bp N-terminal pseudomonas infections [].

Genomic DNA was extracted with The bacterial flagella are strong immunogenic factors phenol-chloroform method as described elsewhere and active or passive immunization with flagellar [17]. ExoA and fliC genes were amplified separately antigens induces antibody production.

Overlapping PCR generating higher level of immune responses. Different was used for construction of exoA-fliC fusion gene cocktails or fusion proteins have been attempted [18].

Purification was performed under denaturing condition using 8 M urea for denaturation. The contamination proteins were washed away from band related to fusion gene was cut and recovered by the column using wash buffer 20 mM sodium using a purification kit MN, Macherey Nagel, phosphate, mM NaCl, 50 mM imidazol, pH 8. The protein was then collected from column by using elution buffer 20 mM sodium phosphate, mM Cloning and sequencing.

Cloning of fusion gene NaCl and mM imidazol, pH 8. Western-blotting was used to sequenced Eurofins MWG Operon, Germany for evaluate the immunological properties of recombinant analysis of the sequence integrity. Then, the reactivity of recombinant protein E. Then, the pET22b- patients with P. In an Hospital Tabriz. The P. HRP-conjugated anti-rabbit IgG for further four hours. For expressed protein solubility analysis, E. After 18 hours, it was gene from P.

Recombinant exoA-fliC fusion protein was expressed as a kDa protein in high concentration. Western-blotting results. Western-blotting analysis showed that recombinant exoA-fliC was reacted with antibody against native exoA antibody Fig. This finding verified the correct conformational structure of recombinant protein produced in E.

Analysis for reactivity of recombinant fusion protein to sera from Fig. Electrophoresis of overlapping PCR product on patients with P. Recombinant expression of exoA-flagellin fusion protein. For high-level expression of exoA-fliC fusion protein in E. Induction of E. Lane 1, standard protein size 1 2 3 marker; lane 2, pellet of un-induced bacteria; lane 3, pellet of IPTG-induced bacteria and lane 4, purified-recombinant fusion protein. Therefore, the management of P.

Nowadays, development of alternative methods for treatment of P. Various new Fig. Electrophoresis of digestion product. Lane 1 and 2, approaches have been investigated to combat pseudo- recombinant vector pET22b-exo-fliC digested whit BamhI and monas infection.

For example, it has been shown that deletion of Glu in the toxic domain of exoA abolishes the toxicity of pseudomonas exoA [28]. In our study, non-toxic exoA with suitable immunogenic properties was obtained by deletion of enzymatic 70 kDa domain. In the recombinant method, not only production of large amounts of fliC is possible but also selection of conserved and immunogenic domains and deleting of unnecessary domains are feasible.