Search results for " Affinity"

Production of biologically active recombinant avidin in baculovirus-infected insect cells

1997

Abstract An efficient lepidopteran insect cell system was established for the expression of a recombinant form of chicken egg-white avidin. The gene product was obtained in both secreted and intracellular forms, and biologically active recombinant avidin was isolated using affinity chromatography on an iminobiotin–agarose column. Similar to the known quaternary structure of the native egg-white protein, the purified recombinant protein was glycosylated and assembled mainly into tetramers. Like native avidin, the recombinant tetramer also exhibited a high level of thermostability, and was further stabilized upon binding biotin. The biotin-binding and structural properties of the recombinant …

Thin-layer affinity chromatography in analysis of protein-ligand affinity.

1996

The human brain hexacoordinated neuroglobin three-dimensional structure

2004

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O2 supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a six-coordinated heme. O2 and CO bind to the heme-iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold, adapted to host the reversible bis-histidyl heme complex, and an elongated protein matrix cavity, held to facilitate O2 diffusion to the heme. The structure of neuroglobin suggests that the classical globin fold is endowed w…

Proteins participating to the post-transcriptional regulation of the mitochondrial cytochrome c oxidase subunit IV via elements located in the 3′UTR

2009

Abstract In developing rat brain cytochrome c oxidase subunit IV (COXIV) expression is also regulated at post-transcriptional level and two 3′UTR-COXIV RNA-binding factors have been identified. Here, we report the enrichment and identification of the factors from just born rat brains by affinity chromatography of biotinylated 3′UTR-COXIV RNA–protein complexes on streptavidin-conjugated paramagnetic particles. We successfully isolated two main proteins of about 86 and 42 kDa, whose sequences were highly attributable to Hsp90 and Actin. The purified proteins maintain RNA-binding ability and specificity for COXIV messenger and, interacting with the 3′UTR, then could negatively modulate mRNA tr…

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

2016

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub…

Bohr-effect and buffering capacity of hemocyanin from the tarantula E. californicum.

2003

A previous report showed that binding of oxygen to the 24-meric hemocyanin from E. californicum does not correlate linearly with the release of protons as known from hemoglobin. However, this unusual complex phenomenological observation could not be explained at that time. Here, I present a full analysis of the thermodynamic coupling between protons and oxygen for the 24-meric tarantula hemocyanin in Ringer-solution based on the Nested-MWC-model. A strategy is presented which allows to reduce the number of free parameters when fitting the model to the data by including explicitly the equilibrium constants for binding of protons to the different conformations. The results show that the Neste…

Enantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral select…

2007

The present paper deals with the enantiomeric separation of six antihistaminic enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and HSA plug length (SPL) was carried out since there are interactions between variables that could not be considered in an univariate optimization. The estimated and experimental resolution values obtained for antihistaminic enantiomers varied from 1.13 (for orphenadrine) to 2.15 (for brompheniramine). The optimum experimental conditions for ena…

Adsorption of gold clusters on metal-supported MgO: Correlation to electron affinity of gold

2007

Adsorption of ${\mathrm{Au}}_{N}$ clusters $(N=1\char21{}6)$ on a bulk MgO(001) surface and on an ultrathin (3 ML) MgO(001) film supported by Mo metal is investigated via density-functional theory calculations. Comparison of the two substrates unambiguously shows that the gold clusters adsorbed on $\mathrm{Mg}\mathrm{O}∕\mathrm{Mo}$ turn into singly charged cluster anions ${\mathrm{Au}}_{N}^{\ensuremath{\delta}}$, $\ensuremath{\delta}\ensuremath{\approx}\ensuremath{-}1$. Their structures and internal charging patterns are analogous to singly charged, planar, gas-phase cluster anions ${\mathrm{Au}}_{N}^{\ensuremath{-}}$. The adsorption energy has prominent size-dependent odd-even oscillation…

Adhesion of 8701-BC breast cancer cells to type V collagen and 67 kDa receptor

1992

Ductal infiltration carcinomas (d.i.c.) of the breast are potentially highly metastatic tumours, associated with drastic alterations of the architecture and molecular composition of the extracellular matrix at the tumour-host interface. 8701-BC, a recently characterized cell line, isolated from primary d.i.c., was used to study different aspects of tumor cell-substratum interactions. Since type V collagen deposition is augmented in d.i.c. we have examined the ability of 8701-BC cells to interact with this collagen species. We have found that cell binding to type V collagen was mediated by protein homologous to the 67 kDa laminin receptor (67-R). This conclusion is substantiated by the follo…

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase fromCandida albicans

1999

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue si…