Dye-ligand Chromatography Research Articles

High-Throughput Screening of Dye-Ligands for Chromatography.

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. The choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices serve as a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose the best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lie in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

Synthesis and Evaluation of Dye-Ligand Affinity Adsorbents for Protein Purification.

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 toward a large number of proteins. They are commercially available, inexpensive, stable and can easily be immobilized. Significant factors that contribute to the successful operation of a dye-ligand chromatography include matrix type, dye-ligand density, adsorption along with elution conditions and flow rate. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a givendye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris on Cibacron Blue 3GA-Sepharose affinity adsorbent is given as an example.

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.

Isolation and characterization of recombinant murine Wnt3a

Wnt proteins are a family of morphogens that possess potent biological activity. Structure–function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt–cyclodextrin complexes may facilitate structure–activity studies in the absence of adverse detergent effects.

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD − phenotype in C. glutamicum: the issue of reversibility re-examined

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.

Remarkable stability of the proton translocating F 1F O-ATP synthase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

For functional characterization, we isolated the F 1F O-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F 1F O subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F 1F O into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 °C, but even at 95 °C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F 1F O-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.

A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD + and NADP +, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG 4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at ≤180 g MAb/L of resin with the PA eluate, reduced BSA (1–2 log), host cell protein (HCP; 2–3 log), MAb oligomer (31–85%), fragment (from ∼0.8% to <0.1%), and other undesired MAb species. Purity, as measured by non-reducing (NR) SDS-PAGE, was improved 33–85%, to 92–99.5% overall (>99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was ≤1 ng/mg in MAb preparations purified through the three column process and then concentrated and buffer exchanged into the appropriate buffer using tangential flow filtration (TFF).

The influence of bakers’ yeast cells on protein adsorption performance in dye-ligand expanded bed chromatography

The influence of whole yeast cells (0–15% w/v) on the protein adsorption performance in dye-ligand chromatography was explored. The adsorption of a model protein, bovine serum albumin (BSA), was selected to demonstrate this approach. The UpFront adsorbent (ρ=1.5 g/cm3) derivatised with Cibacron Blue 3GA and a commercially available expanded bed column (20 mm i.d.) from UpFront Chromatography, Denmark, were employed in the batch binding and expanded bed operation. The BSA binding capacity was demonstrated to not be adversely affected by the presence of yeast cells. The dynamic binding capacity of BSA at aC/C 0=0..1 biomass concentration of 5, 10, 15% w/v were 9, 8, and 7.5 mg/mL of settled adsorbent, respectively.

N-acetyl glutamate kinase from Daucus carota suspension cultures: embryogenic expression profile, purification and characterization

In Daucus carota, N-acetylglutamate-5-phosphotransferase (NAGK; E.C. 2.7.2.8) specific activity was shown to correlate with the progression of somatic embryogenesis and was highest in the latter stages, where growth was most rapid. The enzyme was subsequently purified greater than 1200-fold using heat treatment, ammonium sulfate fractionation, gel filtration, anion exchange and dye ligand chromatography. Carrot NAGK was shown to have a subunit molecular weight of 31 kDa and form a hexamer. The Kms for NAG and ATP are 5.24 and 2.11 mM, respectively. Arginine (Arg) is a K-type allosteric inhibitor of the enzyme, and Hill coefficients in the order of 5 in the presence of Arg suggest that the enzyme is highly cooperative. D. carota NAGK does not bind to Arabidopsis thaliana PII affinity columns, nor does the A. thaliana PII increase NAGK specific activity, indicating its cellular location is probably different.

Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: A comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; ρ = 2.65 g ml −1) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 l h −1) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2 IU mg −1, after a single step elution with 0.15 M NaCl. The generic application of this approach has been evaluated.

A Novel Method for the Separation and Purification of Human Serum Acid Alpha-1-Glycoprotein. Liquid Chromatographic and Mass Spectrometric Investigation of Tryptic Fragments

Analytical ion-exchange chromatography resulted in significantly elevated human serum acid alpha-1-glycoprotein (AGP) levels in cancer patients (153–194 mgdL−1) and in acute inflammation (161 mgdL−1) compared to healthy individuals (58.5 ± 17.4 mgdL−1) and non-tumorous diseases (61.6 ± 5.0 mgdL−1). A novel method of sample preparation and dye-ligand affinity chromatography was worked out and applied to the fast and biocompatible isolation and purification of AGP. Solvent extraction and precipitation provided nearly 100% recovery of serum AGP, while affinity chromatography using a new generation of dye-ligand phase (Toyopearl AF-Blue) resulted in AGP with a total yield over 85%. Tryptic fragments of AGP were separated by reversed phase liquid chromatography (RP-HPLC) and identified either with on-line ESI-MS, or off-line with PNGase F digestion and NH2-phase HPLC. Off-line analysis provided further data on the distribution of antennary oligosaccharide structures. Predominance of bi- and triantennary chains was shown in human serum AGP (38 and 30%, respectively), while significant elevation in the relative proportion of tetraantennary compounds (9.6 vs 15.0%) and a 2-3 fold increase in the total amount of fucose containing structures were observed in cancer patients compared to the healthy controls.

Purification of wheat flour high-Mr glutenin subunits by Reactive Red 120-Agarose and Reactive Yellow 86-Agarose resins

Abstract Two reactive textile dyes were employed to purify some high-Mr glutenin subunit mixtures by dye-ligand chromatography. In particular, the use of the triazine dye Reactive Red 120 coupled to Agarose bed provided an efficient method to purify high-Mr glutenin subunit mixtures, showing high binding capacity and high selectivity for these proteins and assuring a recovery of about 80%. On the other hand, the Reactive Yellow 86 had weaker interaction and a lower selectivity than Red 120 with a recovery of about 60%. However, this dye allowed the separation of some subunits that were not separated by reactive Red 120.

Purification and characterization of mannitol dehydrogenase and identification of the corresponding cDNA from the head blight fungus, Gibberella zeae ( Fusarium graminearum)

The mannitol-2-dehydrogenase (MtDH) from Gibberella zeae was purified and the corresponding cDNA identified. Purification of MtDH was accomplished using a combination of ammonium sulfate fractionation, anion exchange and dye-ligand chromatography. Final purification was achieved following electroelution from a native gel. Molecular mass determination based on SDS–PAGE indicated that the denatured protein was 29 kDa. Native protein mass was determined to be 110 kDa using gel permeation chromatography, indicating a tetrameric form. The pH optima for mannitol oxidation and fructose reductase activities were 9.0, and 7.0, respectively. Activity with sorbitol as the substrate was 21% of activity with mannitol. Kinetic parameters were determined by direct-linear plots of enzyme activity vs. substrate concentrations. Fructose concentrations above 600 mM and NADPH concentrations above 0.3 mM caused substrate inhibition. Comparisons of predicted amino acid sequences of several fungal MtDHs indicated high conservation within the phyla. A possible role for MtDH in generation of turgor pressure for forcible ascospore discharge is discussed.

Purification of Wheat Flour High- Mr Glutenin Subunits by Dye-ligand Chromatography

Abstract Here is reported the first successful attempt of high- Mr glutenin subunits separation by dye-ligand chromatography of Alisei 1 (containing 1Bx7, 1Dx2+1Dy12 subunits), Alisei 2 (containing 1Bx7 and 1Dy12 subunits), and WRU 6979 (containing 1Dx5+1Dy10 subunits), flours, using Cibacron Blue F3GA-Sepharose CL6B as resin. High- Mr glutenin subunits interacted strongly with the Cibacron Blue F3GA ligand and their separation, together with high recovery, was successful by using sodium dodecyl sulphate as eluting agent. Experimental evidence and amino acid sequences of high- Mr glutenin subunits suggested a prevalent electrostatic interaction between high-Mr glutenin subunits and the Cibacron Blue F3GA dye.

6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme.

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.

Binding of 5-(2′-carboxyphenyl)azoquinolin-8-ol to bovine serum albumin: a spectroscopic study

Dye–protein interactions are of immense importance in dye–ligand chromatography of protein purification. In this type of interactions, the structure of the dye molecules has a significant role. However, studies on the structure of these ligands are scanty. Therefore, we have spectroscopically investigated interactions of three 5-(aryl)azoquinolin-8-ol derivatives, which could be used as potent chelate forming agents, with bovine serum albumin (BSA). Among these, the carboxy derivative, 5-(2′-carboxyphenyl)azoquinolin-8-ol (CPAQ) has been selected for resonance Raman study. It has been shown that BSA has six independent binding sites for CPAQ at pH 7.2, the binding constant being 6.2×10 3 M −1. Assignments of Raman modes of bound CPAQ are also presented. It has also been shown that bound CPAQ exists exclusively in hydrazone form. Results further demonstrate that the azo group nitrogen adjacent to the phenyl ring probably participated in the formation of a BSA–CPAQ complex.

The simulated purification of an enzyme as a ‘dry‘ practical within an introductory course of biochemistry**

This article describes a 'dry-laboratory' practical in which the multi-step purification of an enzyme is simulated. It has been devised to be implemented with beginner undergraduates taking an introductory course in biochemistry, with the aim of giving them a glimpse of the intensity of effort involved in complex, research-oriented experiments. The purification steps simulated are: preparation of a liver soluble extract, ammonium sulfate fractionation, gel filtration, ion-exchange and dye-ligand affinity chromatography. Before the simulation, our students are familiar with the preparative and analytical techniques involved, through short 'cookbook' laboratory experiments and, in some cases, ad hoc demonstrations. For the simulation, the students are given detailed protocols of the preparative and analytical experiments, and the raw numeric or graphical data obtained. They have to perform the calculations and graphing necessary to produce a purification table. The 27-page Student Booklet needed to implement the practical is offered by the authors to interested teachers, as a printable electronic file.

Heterogeneity of 11β-hydroxysteroid dehydrogenase type 1/microsomal carbonyl reductase (11β-HSD/CR) in guinea pig tissues. Purification of the liver form suggests modification in the cosubstrate binding site

11β-hydroxysteroid dehydrogenase (11β-HSD) and xenobiotic carbonyl reductase activities were determined in guinea pig tissue microsomes. The data indicate the presence of a NADP(H) dependent form, distinct from the known type I isozyme. Purification of 11β-HSD-1 from liver microsomes resulted in two distinct peaks, resolved by dye–ligand chromatography, indicating differences in the cosubstrate binding site. Immunoblot analysis using anti 11β-HSD-1 antibodies reveals the presence of similar structural determinants between the enzyme forms. Both have an apparent molecular mass of 32 kDa, suggesting protein modifications occurring in the type 1 isozyme which account for the differences in chromatographic behaviour.

Dye-ligand chromatographic purification of intact multisubunit membrane protein complexes: application to the chloroplast H+-FoF1-ATP synthase

n-Dodecyl-β-D-maltoside was used as a detergent to solubilize the ammonium sulphate precipitate of chloroplast FOF1-ATP synthase, which was purified further by dye-ligand chromatography. Upon reconstitution of the purified protein complex into phosphatidylcholine/phosphatidic acid liposomes, ATP synthesis, driven by an artificial ∆pH/∆ψ, was observed. The highest activity was achieved with ATP synthase solubilized in n-dodecyl-β-D-maltoside followed by chromatography with Red 120 dye. The optimal dye for purification with CHAPS was Green 5. All known subunits were present in the monodisperse proton-translocating ATP synthase preparation obtained from chloroplasts.

Dye-ligand chromatographic purification of intact multisubunit membrane protein complexes: application to the chloroplast H+-F0F1-ATP synthase

n-Dodecyl-beta-D-maltoside was used as a detergent to solubilize the ammonium sulphate precipitate of chloroplast F(O)F(1)-ATP synthase, which was purified further by dye-ligand chromatography. Upon reconstitution of the purified protein complex into phosphatidylcholine/phosphatidic acid liposomes, ATP synthesis, driven by an artificial DeltapH/Deltapsi, was observed. The highest activity was achieved with ATP synthase solubilized in n-dodecyl-beta-D-maltoside followed by chromatography with Red 120 dye. The optimal dye for purification with CHAPS was Green 5. All known subunits were present in the monodisperse proton-translocating ATP synthase preparation obtained from chloroplasts.