Large-scale Dye-ligand Chromatography

Scawen, Michael D.; Atkinson, Tony

doi:10.1007/978-1-349-06582-0_4

Cited by 12 publications

(1 citation statement)

References 177 publications

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“…Dye−ligand adsorbents have been used in laboratory‐ and large‐scale protein/enzyme chromatographic purifications (1). In most of these processes, Triazine dyes such as Cibacron Blue are used in place of natural biological molecules (2).…”

Section: Introductionmentioning

confidence: 99%

Dye Affinity Adsorbent Replacement Optimization

et al. 1998

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A method to determine the optimal replacement time for dye affinity adsorbents used in protein purification processes that are subjected to severe regeneration conditions has been developed. To demonstrate the utility of the method, an experimental fixed-bed decay model was employed to determine the optimum number of cycles for the adsorbent replacement. This number is a function of the column regeneration frequency and of the capital and operation costs. The implications of the results on the design and operation of dye-ligand chromatographic processes are discussed.

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Section: Introductionmentioning

confidence: 99%

Dye Affinity Adsorbent Replacement Optimization

et al. 1998

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Affinity Chromatography and its Applications in Large‐Scale Separations

Goward¹

1994

Process Scale Liquid Chromatography

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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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Large-scale Dye-ligand Chromatography

Cited by 12 publications

References 177 publications

Dye Affinity Adsorbent Replacement Optimization

Dye Affinity Adsorbent Replacement Optimization

Affinity Chromatography and its Applications in Large‐Scale Separations

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

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