Improved Refolding of an Immobilized Fusion Protein

Stempfer, Günter; Höll-Neugebauer, Bärbel; Rudolph, Rainer

doi:10.1038/nbt0396-329

Cited by 146 publications

(84 citation statements)

References 15 publications

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“…Whether an unfolded polypeptide takes a native conformation or forms random aggregations should be delicately balanced between hydrophobic and ionic forces (Stempfer et al 1996;Hanson and Gellman 1998). The presence of salts decreases ionic interactions and subsequently increases hydrophobic association of unfolded polypeptides.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry

Yamashita

Okada

2005

J Histochem Cytochem.

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S U M M A R YIn this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl 2 , then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter-and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl 2 showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of ␤ -actin (monomer and oligomers) and fibronectin were present in extracts from deparaffinized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes.

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Section: Discussionmentioning

confidence: 99%

Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry

Yamashita

Okada

2005

J Histochem Cytochem.

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“…At present, only histidine and arginine tags have been found to be suitable for this process, because they maintain matrix binding ability under denaturing conditions (73,189). Recently, Berdichevsky et al (17) demonstrated that a CBM (C. thermocellum) can be used as the attachment support for matrix-assisted refolding VOL.…”

Section: Bioprocessingmentioning

confidence: 99%

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

Shoseyov

Shani

Levy

2006

Microbiol Mol Biol Rev

498

347

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SUMMARY Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular “Velcro” for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications.

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“…-448)-After a first washing of the pellet in 1 M urea which significantly reduced the amount of contaminant bacterial proteins (SDS-PAGE, not shown), the recombinant protein from the insoluble fraction was solubilized in 6 M urea. Based on previous refolding assays using matrixbound proteins (41,42), the refolding of His tag-␣ 5 -(229 -448) was carried out with the denatured protein immobilized on the Ni-NTA affinity matrix (see "Experimental Procedures"). The efficiency of protein refolding was dependent on different parameters that were optimized as follows: (i) the recovery yield is strongly dependent on KCl concentration, as shown in Fig.…”

Section: Cloning and Expression Of ␣ 5 -(229 -448)-two Differentmentioning

confidence: 99%

The Cation-binding Domain from the α Subunit of Integrin α5β1 Is a Minimal Domain for Fibronectin Recognition

Banères¹,

Roquet²,

Green³

et al. 1998

Journal of Biological Chemistry

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The cation-binding domain from the ␣ subunit of human integrin ␣ 5 ␤ 1 was produced as a recombinant protein, ␣ 5 -(229 -448). This protein displays a well defined fold with a content of 30 -35% ␣-helix and 20 -25% ␤-strand, based on circular dichroism. The binding of Ca 2؉ or Mg 2؉ to ␣ 5 -(229 -448) results in a biphasic conformational rearrangement consistent with the occurrence of two classes of cation-binding sites differing by their affinities. The two classes of sites are located in two conformationally independent lobes, as established by a parallel study of two recombinant half-domains (Nand C-terminal) that also adopt stable folds. Upon saturation with divalent cations, ␣ 5 -(229 -448) binds an ArgGly-Asp (RGD)-containing fibronectin ligand to form a 1:1 complex. Complex formation is associated with a specific conformational adaptation of the ligand, suggesting an induced fit mechanism. In contrast, neither of the half-domains is competent for ligand binding. The ␣ 5 -(229 -448)-fibronectin complex is dissociated in the presence of an RGD peptide, as well as of a simple carboxylic acid, suggesting that the RGD aspartyl carboxylate is an essential element that directly interacts with the ␣ 5 cation-binding domain. Integrins (IN)1 are a family of structurally and functionally related adhesion receptors that participate in cell-cell and cellextracellular matrix interactions (1, 2). All integrins are heterodimers of non-covalently associated ␣ and ␤ subunits. In general, ligand binding occurs through recognition by the integrin of a short amino acid sequence from the ligand (3). The prototype for these integrin-binding sites is the Arg-Gly-Asp (RGD) sequence (4) that is present in fibronectin, fibrinogen, vitronectin, and other adhesive proteins (5). Divalent cations are essential for integrin function (6). Both integrin ␣ and ␤ subunits have been implicated in cation and ligand binding (7), although the precise nature of the ligand recognition site on integrins remains elusive. The ␤ subunits are characterized by a conserved domain in their N-terminal regions that contributes to cation as well as ligand binding (8 -10). The integrin ␣ subunits are characterized by the presence of seven N-terminal repeats that encompass nearly half of the subunit residues (5). Three to four of these repeats (IV or V to VII) display sequences that resemble the EF-hand consensus sequence found in various divalent cation-binding proteins (11). However, the integrin EF-hands systematically lack an acidic residue at their relative positions 12, a highly invariant Glu residue in the typical EF-hands, that is replaced by a non-polar residue. It has been hypothesized that the integrin EF-hands are responsible for the cation binding properties of the integrin ␣ subunits (11). In agreement with this hypothesis, a soluble recombinant fragment that encompasses the four EF-hand type sequences from the ␣ subunit of integrin ␣ IIb ␤ 3 displays four Ca 2ϩ -binding sites distributed in two classes differing by their affinities (12). Moreo...

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Improved Refolding of an Immobilized Fusion Protein

Cited by 146 publications

References 15 publications

Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry

Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

The Cation-binding Domain from the α Subunit of Integrin α5β1 Is a Minimal Domain for Fibronectin Recognition

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