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1.
Anal Biochem ; 423(2): 202-9, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22342623

RESUMEN

This study presents a methodology for covalent attachment of hydrophobic peptidic ligands to hydrophilic chromatographic matrices with improved coupling efficiency. Preconcentration was introduced through the use of polyethylene glycol (PEG)-based crosslinkers. Immobilization of model hydrophobic peptide pep12 (ITLISSEGYVSS) to hydrophilic silica-amine matrix was investigated in the absence/presence of PEG-based linker. The effect of linker densities 14.2, 27.6, and 56.4 µmol/g beads on coupling efficiency was investigated. Whereas a ligand coupling efficiency of 67% was obtained in the absence of the linker, incorporating PEG-based linker at low densities allowed a 30% increase in the coupling efficiency. Although the heterobifunctional crosslinker, maleimide-PEG-NHS (N-hydroxysuccinimide) ester, can be used to couple thiol-bearing ligands to amine-functionalized matrices, no method is available for quenching free amine moieties on the matrix after ligand immobilization. The efficacy of acylating agents, acetyl chloride and oxalyl chloride, in blocking free amine groups when immobilizing the model peptide pep14 (CITLISSEGYVSSK) to silica-amine matrix using maleimide-PEG-NHS ester crosslinker was investigated. Because oxalyl chloride was nonreactive to maleimides, it allowed successful coupling of pep14 to the maleimide termini of the linkers. Adsorption studies between pep14-immobilized microspheres and human immunoglobulin M (hIgM) suggested retention of ligand activity and a 95% decrease in nonspecific binding of proteins to the matrix.


Asunto(s)
Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Péptidos/química , Adsorción , Secuencia de Aminoácidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Inmunoglobulina M/química , Inmunoglobulina M/metabolismo , Ligandos , Maleimidas/química , Péptidos/metabolismo , Polietilenglicoles/química , Dióxido de Silicio/química , Succinimidas/química
2.
Biotechnol Adv ; 29(6): 840-9, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21762771

RESUMEN

Extensive research in the past two decades has led to the realization of Immunoglobulin-M (IgM) as a potential therapeutic and diagnostic agent. In order to fully exploit the potential of IgM, large quantities, in a highly pure and active form, must be available at low cost for performing clinical trials, characterization studies and quantitative-structure activity analyses. The complex physico-chemical properties, in particular its large size and labile nature renders downstream purification of IgM difficult. This review discusses the limitations and challenges associated with the current IgM purification strategies and proposes future directions for research. The uniqueness of affinity chromatography, specifically biomimetic affinity chromatography for protein purification is highlighted and its potential for IgM purification is discussed.


Asunto(s)
Biotecnología/métodos , Cromatografía de Afinidad/métodos , Inmunoglobulina M/aislamiento & purificación , Humanos
3.
Anal Biochem ; 386(2): 194-216, 2009 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-19133223

RESUMEN

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.


Asunto(s)
Técnicas Biosensibles/métodos , Proteínas/análisis , Anticuerpos Catalíticos/análisis , Benchmarking , Sitios de Unión , Técnicas Biosensibles/estadística & datos numéricos , Glutatión Transferasa/análisis , Cinética , Ligandos
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