Search results for "Affinity chromatography"

F-type lectin from serum of the Antarctic teleost fish Trematomus bernacchii (Boulenger, 1902): Purification, structural characterization, and bacter…

2021

Abstract The increasing availability of sequenced genomes has enabled a deeper understanding of the complexity of fish lectin repertoires involved in early development and immune recognition. The teleost fucose-type lectin (FTL) family includes proteins that preferentially bind fucose and display tandemly arrayed carbohydrate-recognition domains (CRDs) or are found in mosaic combinations with other domains. They function as opsonins, promoting phagocytosis and the clearance of microbial pathogens. The Antarctic fish Trematomus bernacchii is a Perciforme living at extremely low temperatures (−1.68 °C) which is considered a model for studying adaptability to the variability of environmental w…

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

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus

2016

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine- agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino- acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, 
 including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemag…

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…

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…

Site specificity of pea histone acetyltransferase B in vitro.

1993

Histone acetyltransferase B from pea embryonic axes has been purified approximately 300-fold by a combination of chromatographic procedures, including affinity chromatography on histone-agarose. The enzyme preparation has been used for the in vitro transfer of acetyl groups from [1-14C]acetyl-CoA to non-acetylated pea histone H4. Up to three acetyl groups can be introduced into the histone. The resulting mono-, di-, and triacetylated H4 isoforms were separated and sequenced to determine the acetylated sites. Only sites 5, 12, and 16 were used by histone acetyltransferase B, but no clear preference among them was observed. The absence of modification of other potentially acetylatable sites i…

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28.

1987

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannos…

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum.

2007

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose-agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 degrees C, retained partial activity by treatment at 70 degrees C, and was fully inactivated at 90 degrees C. On western blot anal…

A serum fucolectin isolated and characterized from sea bass Dicentrarchus labrax

2001

A lectin specific for fucose and galactose was isolated by affinity chromatography on Sepharose CL-6B from the serum of Dicentrarchus labrax. The hemagglutinating activity against rabbit erythrocytes was calcium-independent, and reached its maximum at 37 degrees C. Two protein components were found in the hemagglutinating fractions eluted from the Sepharose column. Only the 34 kDa component (DLL2) eluted from the polyacrylamide gels (SDS-PAGE) showed agglutinating activity against rabbit erythrocytes. SDS-PAGE, in non-reducing conditions, revealed a single 66 kDa protein that reacted with antibodies to the 34 kDa component. Therefore, a dimeric structure stabilized by disulfide bonds can be…