Engineering DNA and Protein Chimeras Utilizing Coding Sequences of Restriction Sites

Sinha, Dipali; Bakhshi, Meenakshi R.; Vora, Renu; Kirby, Edward P.; Budzynski, Andrei Z.

doi:10.1006/abio.1996.0277

Cited by 6 publications

(3 citation statements)

References 1 publication

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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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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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“…Soluble scFv [EBB3] fragments were isolated in the cytoplasmic compartment using IMAC in packed bed. The purification of insoluble scFv[EBB3] (inclusion bodies) requires a solubilization step [17] before capture by IMAC then followed by a refolding procedure performed by buffer exchange onto the column for isolation of scFv in the native form [18][19][20][21][22][23]. Biological activity was assessed for all IMAC purifications by Enzyme Linked Immunosorbent Assay (ELISA), cytometric assays and immunohistochemistry.…”

Section: Introductionmentioning

confidence: 99%

Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin

Robert

Clofent‐Sanchez

Hocquellet

et al. 2006

International Journal of Biological Macromolecules

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“…The refolding process can be performed with different chromatographic methods, size exclusion chromatography [18][19][20][21][22][23][24][25][26], immobilization on gel matrices [27], ion exchange chromatography [28], hydrophobic interaction chromatography [29], immobilized metal affinity chromatography [30][31][32][33][34][35], affinity chromatography [36,37], immobilized liposome chromatography [38].…”

Section: Introductionmentioning

confidence: 99%

Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli

Vincent

Dieryck

Maneta-Peyret

et al. 2004

Journal of Chromatography B

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Engineering DNA and Protein Chimeras Utilizing Coding Sequences of Restriction Sites

Cited by 6 publications

References 1 publication

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

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

Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin

Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli

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