Novel fiber-optic biosensor based on immobilized glutathione S-transferase and sol–gel entrapped bromcresol green for the determination of atrazine

The aim of this work was to investigate, for the first time, the potential of the enzyme glutathione S-transferase I (isoenzyme GST-I) for uses in analytical chemistry. A novel fiber-optic biosensor for the detection and determination of the triazine herbicide atrazine was developed based on maize GST-I expressed in E. coli. The sensing bioactive material was a three-layer mini-sandwich. The enzyme was immobilized on the outer layer that consisted of a hydrophilic polyvinylidenefluoride membrane. This membrane was supported on an inner glass disk by means of an intermediate binder sol–gel layer that incorporated bromcresol green (BCG). The biosensor operated in a static mode at 25 °C and the rate of the enzymatic reaction, using atrazine as a substrate, served as an analytical signal. A calibration curve was obtained for atrazine, with analytically useful concentration range 2.52–125 μM. The sensor detection limit was 0.84 μM. The reproducibility of atrazine sensing was in the order of ±3–5%. The method was successfully applied to the determination of this herbicide in real water samples, without sample preparation steps. Atrazine recovery ranged between 85 and 110%. No interference from other pesticides, such as alachlor and carbaryl was observed in the absence of atrazine. The immobilized enzyme retained about 75% of its original activity after 1 month use. Simply unscrewing the terminal holding ring of the probe and placing a new bioactive sandwich could easily replace a deteriorated mini-sandwich.     

Biomimetic dyes as affinity chromatography tools in enzyme purification

Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins. Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation, the model of the protein and also a number of suitable synthetic ligands which mimic natural biological ligands of the protein. There are several examples of enzyme purifications (trypsin, urokinase, kallikrein, alkaline phosphatase, malate dehydrogenase, formate dehydrogenase, oxaloacetate decarboxylase and lactate dehydrogenase) where synthetic biomimetic dyes have been used successfully as affinity chromatography tools.     

Dye-ligand chromatography for the resolution and purification of restriction endonucleases

The resolution of restriction endonucleases from the same microorganism is conventionally achieved by lengthy fractionation protocols. We now report effective single-step procedures that exploit dye-ligand chromatography for the resolution and purification of restriction enzymes. After suitable initial screening, we demonstrated that resolution of two restriction activites can be achieved in one chromatographic step, and further purification can subsequently be effected using selected dye-adsorbents. Accordingly, we resolved in one step, Hpa I from Hpa II, Hind II from Hind III, and Sac I from Sac II. Furthermore, a three-step Chromatographic procedure has been developed to purify EcoRV suitable for commercial exploitation, as judged by the “overdigestion” and “cut-ligate-recut” quality control tests.     

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.     

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.     

Affinity chromatography matures as bioinformatic and combinatorial tools develop

Affinity chromatography has the reputation of a more expensive and less robust than other types of liquid chromatography. Furthermore, the technique is considered to stand a modest chance of large-scale purification of proteinaceous pharmaceuticals. This perception is changing because of the pressure for quality protein therapeutics, and the realization that higher returns can be expected when ensuring fewer purification steps and increased product recovery. These developments necessitated a rethinking of the protein purification processes and restored the interest for affinity chromatography. This liquid chromatography technique is designed to offer high specificity, being able to safely guide protein manufactures to successfully cope with the aforementioned challenges. Affinity ligands are distinguished into synthetic and biological. These can be generated by rational design or selected from ligand libraries. Synthetic ligands are generated by three methods. The rational method features the functional approach and the structural template approach. The combinatorial method relies on the selection of ligands from a library of synthetic ligands synthesized randomly. The combined method employs both methods, that is, the ligand is selected from an intentionally biased library based on a rationally designed ligand. Biological ligands are selected by employing high-throughput biological techniques, e.g. phage- and ribosome-display for peptide and microprotein ligands, in addition to SELEX for oligonucleotide ligands. Synthetic mimodyes and chimaeric dye-ligands are usually designed by rational approaches and comprise a chloro-triazinlyl scaffold. The latter substituted with various amino acids, carbocyclic, and heterocyclic groups, generates libraries from which synthetic ligands can be selected. A 'lead' compound may help to generating a 'focused' or 'biased' library. This can be designed by various approaches, e.g.: (i) using a natural ligand-protein complex as a template; (ii) applying the principle of complementarity to exposed residues of the protein structure; and (iii) mimicking directly a natural biological recognition interaction. Affinity ligands, based on the peptide structure, can be peptides, peptide-mimetic derivatives (<30 monomers) and microproteins (e.g. 25-200 monomers). Microprotein ligands are selected from biological libraries constructed of variegated protein domains, e.g. minibody, Kunitz, tendamist, cellulose-binding domain, scFv, Cytb562, zinc-finger, SpA-analogue (Z-domain).     

Lock-and-key motif as a concept for designing affinity adsorbents for protein purification

The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.     

Designed chimaeric galactosyl-mimodye ligands for the purification of Pseudomonas fluorescens beta-galactose dehydrogenase

Two chimaeric galactosyl-mimodye ligands were designed and applied to the purification of Pseudomonas fluorescens galactose dehydrogenase (GaDH). The chimaeric affinity ligands comprised a triazine ring on which were anchored: (i) an anthraquinone moiety that pseudomimics the adenine part of NAD+, (ii) a galactosyl-mimetic moiety (D-galactosamine for ligand BM1 or shikimate for ligand BM2), bearing an aliphatic 'linker', that mimics the natural substrate galactose, and (iii) a long hydrophilic 'spacer'. The mimodye-ligands were immobilised to 1,1-carbonyldiimidazole-activated agarose chromatography support, via the spacer's terminal amino-group, to produce the respective mimodye adsorbents. Both immobilized mimodyes successfully bound P. fluorescens GaDH but failed to bind the enzyme from rabbit muscle. Adsorbent BM1 bound GaDH from green peas and Baker's yeast, but adsorbent BM2 failed to do so. The mimodye-ligand comprising D(+)-galactosamine (BM1), compared to BM2, exhibited higher purifying ability and enzyme recovery for P. fluorescens GaDH. The dissociation constants (KD) of BM1 and BM2 for P. fluorescens GaDH were determined by analytical affinity chromatography to be 5.9 microM and 15.4 microM, respectively. The binding capacities of adsorbents BM1 and BM2 were 18 U/mg adsorbent and 6 U/mg adsorbent, respectively. Adsorbents BM1 and BM2 were integrated in two different protocols for the purification P. fluorescens GaDH. Both protocols comprised as a common first step DEAE anion-exchange chromatography, with a second step of affinity chromatography on BM1 or BM2, respectively. The purified GaDH obtained from the protocols using BM1 and BM2 showed specific activities equal to 1077 and 854 U/mg, respectively. The former is the highest reported so far and the enzyme appeared as a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.     

Monosized adsorbents for high-performance affinity chromatography. Application to the purification of calf intestinal alkaline phosphatase and human urine urokinase

Affinity adsorbents comprising monodisperse spherical synthetic macroporous beads offer the prospect of high-capacity, high-resolution separation of proteins at low operating pressures. Purpose-designed biomimetic dyes were covalently attached to Dynospheres XP-3507 beads and exploited for the purification of calf intestine alkaline phosphatase and human urine urokinase from crude extracts. This study demonstrates that the combination of specifically designed affinity ligands with monosized support materials is a powerful approach to the resolution of proteins by high-performance affinity chromatography.     

Galactosyl-biomimetic dye-ligands for the purification of Dactylium dendroides galactose oxidase

Two anthraquinone galactosyl-biomimetic dye-ligands comprising, as terminal biomimetic moiety, galactose analogues (1-amino-1-deoxy-beta-D-galactose and D(+)-galactosamine) were designed for the enzyme galactose oxidase (GAO), using molecular modelling, synthesized and characterized. The biomimetic ligands were immobilized on agarose beads and the affinity adsorbents, together with a non-biomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their ability to purify GAO from Dactylium dendroides. Both biomimetic adsorbents showed higher purifying ability for GAO compared to the non-biomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising, as terminal biomimetic moiety, 1-amino-1-deoxy-beta-D-galactose (BM1) exhibited the highest purifying ability for GAO. This affinity adsorbent did not bind galactose dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, or glucose oxidase. The dissociation constant (K(D)) of the immobilized BM1 ligand with GAO was found to be equal to 45.8 microM, whereas the binding capacity was equal to 709 U per ml adsorbent. Therefore, the BMI adsorbent was integrated in a facile two-step purification procedure for GAO. The purified enzyme showed a specific activity equal to 2038 U/mg, the highest reported so far, approximately 74% overall recovery and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis.