Biomimetic dyes as affinity chromatography tools in enzyme purification

Clonis, Yannis D.; Labrou, Nikolaos E.; Vp, Kotsira; Mazitsos, C.F; Melissis, Sotirios; Gogolas, G

doi:10.1016/s0021-9673(00)00577-x

Cited by 114 publications

(60 citation statements)

References 30 publications

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“…The encouraging behavior of the AEAd ligand is supported by adsorption studies, which suggests the existence of some degree of specificity between immobilized AEAd and soluble M-MLVH -RT, after perturbation of the respective complex by ATP. The lower enzyme binding capacity of the AEAd adsorbent, compared to the acclaimed higher (by up to 10-fold) for immobilized triazine dyes (26,27), may be counterbalanced by its effective enzyme purifying ability (Figure 2A and Figure 5). Therefore, adsorbent AEAd was justifiably chosen for M-MLVH -RT purification.…”

Section: Discussionmentioning

confidence: 99%

Purification of M-MLVH- RT on a 9-Aminoethyladenine-(1,6-diamine-hexane)-triazine Selected from a Combinatorial Library of dNTP-Mimetic Ligands

Melissis

Papageorgiou

Labrou

et al. 2010

Journal of Chromatographic Science

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Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH -RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH -RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions ( IntroductionRecombinant proteins are used throughout biomedical and biological sciences. However, their widespread application was made possible thanks to the development of simple and effective protein purification processes that are able to produce proteins of certain purity and quantity, as they may be required. In this endeavor, affinity chromatography (1-3) is the most reliable and indispensable partner for preparing pure proteins because it explores the ability of biologically active macromolecules to form specific and reversible complexes with appropriate affinity ligands.The selection and design of affinity ligands has progressed significantly over the years mainly because of knowledge derived from the scientific interaction between X-ray crystallography and bioinformatics as well as defined and combinatorial chemical synthesis and high throughput screening (1-5). We have reported the design and chemical synthesis of a combinatorial library of novel nucleotide-mimetic synthetic ligands (6,7). These ligands were generated following the so-called "structureguided" or "directed" combinatorial method (2), for which one should have enough in-hand structural data for the target protein. The "winner" ligand is selected from a limited library built around a rationally designed "lead" ligand, a notion that is reminiscent of drug design. In the present study, we explore the dNTP-mimetic ligand library for selecting a ligand suitable for affinity chromatography purification of Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH -RT) derived from recombinant Escherichia coli cells. Possible success of this task would lead to the first purification method for a non-tagged (native) reverse transcriptase by synthetic ligand affinity chromatography.Reverse transcriptase (RT) is an essential retroviral enzyme that cat...

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Section: Discussionmentioning

confidence: 99%

Purification of M-MLVH- RT on a 9-Aminoethyladenine-(1,6-diamine-hexane)-triazine Selected from a Combinatorial Library of dNTP-Mimetic Ligands

Melissis

Papageorgiou

Labrou

et al. 2010

Journal of Chromatographic Science

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“…Covalent attachment of the dye can be achieved through nucleophilic displacement of the dye's chlorine atom by hydroxyl groups on the support's surface (Labrou, 2000;Labrou et al, 1995;Labrou, 2002). Chlorotriazine polysulfonated aromatic molecules (triazine dyes) have been used for the purification of albumin, oxidoreductases, decarboxylases, glycolytic enzymes, nucleases, hydroloases, lyases, synthetases, and transferases (Labrou, 2000;Labrou et al, 1995). The main advantages of using dye-ligands and biomimetic dye-ligands are their low cost and resistance to chemical and biological degradation.…”

Section: Ligands Used In Affinity Chromatographymentioning

confidence: 99%

“…The main disadvantage of these synthetic ligands is that the selection process for a particular biomolecule is empirical and requires extensive screening processes during method development. More information on biomimetic dyes can be found in reference (Clonis et al, 2000).…”

Section: Ligands Used In Affinity Chromatographymentioning

confidence: 99%

Affinity Chromatography: Principles and Applications

Magdeldin¹,

Moser²

2012

Affinity Chromatography

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“…The latter issue is tackled using purification strategies, which in some cases may be a long and tedious process, while in other cases it includes just one chromatographic step (Clonis et al, 2000, Porath, 1992, Wilchek et al, 1984, Zeng and Ruckenstein, 1999. Nevertheless, even in the best case scenario this may have a negative economic impact in the final cost of the biocatalyst.…”

Section: Introductionmentioning

confidence: 99%

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

Barbosa

Ortíz

Berenguer-Murcia

et al. 2015

Biotechnology Advances

600

318

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Enzyme immobilization and purification are two steps that are usually required in the development of any industrial biocatalyst. In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies (versus the target enzyme or versus some domains), the development of specific domains with affinity for some specific supports or just to increase the affinity for standard ones (ionic exchangers, silicates) will be revised. We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailormade heterofunctional supports as a tool to immobilize-stabilize-purify some proteins will be discussed in deep, using low concentration of adsorbents groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be a the last part of the review.

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Biomimetic dyes as affinity chromatography tools in enzyme purification

Cited by 114 publications

References 30 publications

Purification of M-MLVH- RT on a 9-Aminoethyladenine-(1,6-diamine-hexane)-triazine Selected from a Combinatorial Library of dNTP-Mimetic Ligands

Purification of M-MLVH- RT on a 9-Aminoethyladenine-(1,6-diamine-hexane)-triazine Selected from a Combinatorial Library of dNTP-Mimetic Ligands

Affinity Chromatography: Principles and Applications

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

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