Interaction of l-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography

Katsos, N.E.; Labrou, Nikolaos E.; Clonis, Yannis D.

doi:10.1016/j.jchromb.2004.04.032

Cited by 18 publications

(5 citation statements)

References 41 publications

(60 reference statements)

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“…For example, by the rational method (Katsos et al, 2004;Labrou and Clonis, 1995) and by the ''structural template approach'' (Li et al, 1998;Labrou et al, 1999;Melissis et al, 2001;Mazitsos et al, 2002). The former is applied when enough structural data for the target protein are not available, whereas the latter is applied when data on the protein structure and on the interaction between the protein and its natural ligands are available.…”

Section: Resultsmentioning

confidence: 99%

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Platis

Maltezos

K‐C.

et al. 2009

J of Molecular Recognition

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Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.

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Section: Resultsmentioning

confidence: 99%

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Platis

Maltezos

K‐C.

et al. 2009

J of Molecular Recognition

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“…The adsorption capacity of the affinity medium was analyzed according to the method reported [18]. In a total volume of 1 ml varying amounts of purified serine proteinase solution (0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, and 2.7 nM), previously dialyzed against 10 mM sodium phosphate/1 mM EDTA/0.05 M NaCl (pH 6.0), were mixed with 2 mg of affinity medium.…”

Section: Adsorption Analysismentioning

confidence: 99%

Affinity purification of serine proteinase from Deinagkistrodon acutus venom

Xin¹,

Dong²,

Wang³

et al. 2007

Journal of Chromatography B

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“…The amount of dye and the reaction time required to effect immobilized dye concentration in the range of 2.0-3.0 μmol dye/g gel depends on the chemical nature of the dye (e.g., dichlorotriazine dyes in general are more reactive than monochlorotriazines; thus less dye and shorter reaction times are required). In the case of biomimetic dyes, the nature of terminal biomimetic moiety (aliphatic or aromatic substituent) infl uences the electrophilicity of the triazine chloride and thus the reaction time [ 8 ].…”

Section: Notesmentioning

confidence: 99%

Synthesis and Application of Dye-Ligand Affinity Adsorbents

Chronopoulou

Labrou

2014

Methods in Molecular Biology

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Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity towards a large number of proteins. They are commercially available, are inexpensive, and can easily be immobilized. Important factors that contribute to the successful operation of a dye-ligand chromatography include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions used in the adsorption and elution stages, and contacting parameters like flow rate and column geometry. In general, with dye-ligand affinity chromatography, the specificity is provided by the adsorption and elution conditions employed in a particular purification, and these must often be worked out by trial and error. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris crude extract on Cibacron Blue 3GA-Sepharose is given as an example.

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Interaction of l-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography

Cited by 18 publications

References 41 publications

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Affinity purification of serine proteinase from Deinagkistrodon acutus venom

Synthesis and Application of Dye-Ligand Affinity Adsorbents

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