Affinity Chromatography

Lowe, Christopher R.; Clonis, Yannis D.

doi:10.1007/978-1-4757-0405-1_9

Cited by 5 publications

(3 citation statements)

References 198 publications

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“…However, our own measurements performed on ECH activated supports made under the conditions these authors described, yielded a low oxirane density of 371 mol/g dried support. The high concentrations of ECH (30%) and alkali (~0.7 M NaOH), coupled with the extended reaction time (21 h) employed by Maharjan et al [24], are documented to result in extensive cross-linking of the support and increased hydrolysis of introduced oxirane functions to yield glycerol groups [27,[31][32][33][34].…”

Section: Manufacture and Characterization Of The Thermocex Adsorbentmentioning

confidence: 99%

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

Müller

Cao

Ewert

et al. 2013

Journal of Chromatography A

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Section: Manufacture and Characterization Of The Thermocex Adsorbentmentioning

confidence: 99%

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

Müller

Cao

Ewert

et al. 2013

Journal of Chromatography A

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“…Immobilized ligand 11/11 was able to withstand cleaning in situ without a concomitant ligand leakage, assessed by its unchanged ligand density throughout the 10 cycles in both sanitization procedures, although the presence of the ligand in the eluate was not monitored. This stability can be attributed to several factors: first, the polymeric support, Sepharose CL-6B is a highly stable cross-linked solid support matrix especially designed by Amersham Pharmacia Biotech for uses in industrial affinity chromatography, with a pH stability between 3 and 13, stability in several organic solvents, urea and guanidine hydrochloride, and amenability to steam sterilization (120 C for 20 min in pH 7.0); secondly, the epoxide linker chemistry exploited in this study comprises stable ether bonds between the linker and Sepharose (Porath et al, 1970;Lowe and Dean, 1974); thirdly, the scaffold molecule, cyanuric chloride forms non-fissile bonds with its substituents, that are highly stable to chemical degradation compared to the fragile peptide bonds in proteins and, finally, the benzylamine substituents linked to the scaffold molecule lack any unstable and fissile bonds, ensuring the integrity of each monomer on the solid support matrix.…”

Section: Measurement Of Binding Constants Between Immobilized Ligand mentioning

confidence: 99%

An artificial receptor for glycoproteins

Gupta

Lowe

2004

J of Molecular Recognition

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We describe the rational design, synthesis and development of a sterilizable biomimetic ligand for the affinity purification of glycoproteins. Based on mimicking the principles of natural carbohydrate recognition, a putative library of 196 glycoprotein-binding synthetic ligands was designed and synthesized on a polymeric support. Ligand 11/11, based on a triazine scaffold and immobilized on a hydrophilic support, was identified as the "lead" ligand. The carbohydrate recognizing the potential of the "lead" ligand was revealed by reduced binding of a periodate oxidized model glycoprotein, and by "sharp" elution profiles achieved with borate buffer eluents. Specific elution and competitive binding experiments determined the monosaccharide specificity of 11/11 in the order mannoside > glucoside > galactoside. The diastereo-selective performance of ligand 11/11 was quantified and reaffirmed by analytical affinity chromatography and (1)H-NMR, in the order galactoside < glucoside < mannoside with binding affinities (K(a), M(-1)) in the 63-214 and 20-83 M(-1) range, respectively. Partition coefficient analysis revealed binding constants towards glycoproteins in the 10(4) M(-1) range, that compared favourably with the affinities of carbohydrate binding lectins for glycoproteins, such as concanavalin A. Molecular modelling studies of ligand 11/11 revealed the formation of a pre-organized apolar "tweezer-like" cavity, containing complementary nitrogenous hydrogen bond donor and acceptor groups that formed selective interactions with the equatorial 3- and 4-hydroxyl groups of saccharides.

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“…The new biomimetic adsorbents (BM) were expected to show increased purifying tography. I 1 .14,18,2 I , 26 Biotechnology and Bioengineering, Vol. 48, Pp.…”

Section: Introductionmentioning

confidence: 99%

Biomimetic‐dye affinity adsorbents for enzyme purification: Application to the one‐step purification of Candida boidinii formate dehydrogenase

Labrou

Karagouni

Clonis

1995

Biotech & Bioengineering

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Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 microM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C).

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Affinity Chromatography

Cited by 5 publications

References 198 publications

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

An artificial receptor for glycoproteins

Biomimetic‐dye affinity adsorbents for enzyme purification: Application to the one‐step purification of Candida boidinii formate dehydrogenase

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