Effects of ligand concentration on protein adsorption in dye-ligand adsorbents

Purification technologies for plant proteins

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® 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°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 ( KD) of the complex between immobilized BM6 and FDH was found to equal 0.05 μM. 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°C). © 1995 John Wiley & Sons, Inc.

Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase.

Dye-ligand chromatography has developed into an important method for large-scale purification of proteins. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers both the ability to interact with a large number of proteins as well as easy immobilization on typical adsorbent matrices. Reactive dyes can bind proteins either by specific interactions at the protein's active site or by a range of non-specific interactions. Divalent metals participate in yet another type of protein-reactive dye interactions which involve the formation of a ternary complex. All of these types of interactions have been exploited in schemes for protein purification. Many factors contribute to the successful operation of a dye-ligand chromatography process. These include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions employed in the adsorption and elution stages, and contacting parameters like flowrate and column geometry. Dye-ligand chromatography has been demonstrated to be suitable for large-scale protein purification due to their high selectivity, stability, and economy. Also, the issue of dye leakage and process validation of large-scale dye-ligand chromatography has been discussed. Reactive dyes have also been applied in high performance liquid affinity chromatographic techniques for protein purification, as well as non-chromatographic techniques including affinity partition, affinity membrane separations, affinity cross-flow filtration, and affinity precipitation.

Protein purification by dye-ligand chromatography

(1988). The Applications of Reactive Dyes in Enzyme and Protein Downstream Processing. Critical Reviews in Biotechnology: Vol. 7, No. 4, pp. 263-279.

The Applications of Reactive Dyes in Enzyme and Protein Downstream Processing

Affinity chromatography can prove an elegant and efficient solution to the problem of purifying individual enzymes from a complex mixture, and is frequently applied to such purifications on a laboratory scale. However, affinity chromatography is rarely employed for the large-scale purification of enzymes, despite the apparent advantages, in terms of rapid processing, high recovery and ease of automation (Janson, 1982) which the technique possesses. Conventional affinity ligands which have been used for the large-scale purification of enzymes fall into two main groups: (1) those which are specific for the desired enzyme, in which case the ligand is generally a substrate, a substrate analogue or an inhibitor of the enzyme; (2) those which will interact with a wide range of enzymes because the ligand is one which is recognised by many enzymes. Such ligands are immobilised nucleotide cofactors such as NAD, 5–-AMP or 2–,5–-ADP (Mosbach, 1974). General ligands can be employed for the separation of similar types of enzymes on the basis of their differing affinities for the ligand, allowing the selection of appropriate adsorption and/or elution conditions.

Large-scale Dye-ligand Chromatography

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

T. Atkinson

Author Tools

Chat about Author

Papers

Large-scale Dye-ligand Chromatography