Affinity chromatography matures as bioinformatic and combinatorial tools develop

Clonis, Yannis D.

doi:10.1016/j.chroma.2005.09.073

Cited by 128 publications

(103 citation statements)

References 163 publications

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“…As the NMR data indicated that the QQ-mutant displays better side chain packing at neutral pH, a QQ-model (YM 4 In the N-cap: R24 (blue) and S27 (orange), the deletion site of R32 is marked by a black ball. In the four internal repeats: Q59, Q62, Q101, Q104, Q143, Q146, Q185, and Q188; the glutamine at position 26 of each repeat is depicted in cyan, the one at position 29 in magenta.…”

Section: Resultsmentioning

confidence: 99%

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

et al. 2012

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A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature-and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5°higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.

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Section: Resultsmentioning

confidence: 99%

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

et al. 2012

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“…The design, synthesis and selection of affinity ligands have progressed rapidly because of the accumulated knowledge from combinatorial chemistry, structural biochemistry and computational chemistry [3,4]. Recently, we reported a combinatorial library of novel nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes, using Pyrococcus furiosus DNA polymerase (Pfu Pol) as a proof-of-principle example [5].…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we reported a combinatorial library of novel nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes, using Pyrococcus furiosus DNA polymerase (Pfu Pol) as a proof-of-principle example [5]. The nucleotide-mimetic synthetic ligands were generated following the 'structure-guided combinatorial method' [3], according to which, the 'winner' ligand is selected from an intentionally biased ligand library based on a rationally designed 'lead' ligand. In the present study we investigate the applicability of the nucleotide-mimetic ligand library to the selection of ligands for the affinity chromatography purification of thermostable Thermus aquaticus DNA polymerase (Taq Pol) from recombinant Escherichia coli cells.…”

Section: Introductionmentioning

confidence: 99%

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One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

Melissis

Labrou

Clonis

2007

Biotechnology Journal

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The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (K D ) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, ~61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities. The introduction of Taq Pol into PCR has caused this method to evolve into a widely used technique for genetic analysis [6][7][8]. Taq Pol belongs to the family of DNA polymerases I (DNA Pol I), as does the E. coli DNA polymerase I [9]. Because of its high turnover number [10], lack of proofreading activity (3'-5' exonuclease activity) [11], high temperature optimum (72-74°C), and ability to incorporate 7-deaza-3-deoxyquanosine, Taq Pol has been used extensively in DNA sequencing and other molecular biology techniques [12,13]. This enzyme holds academic interest and commercial importance and was, therefore, chosen for further evaluation of the ligand library. A successful affinity ligand selected from the library should be able to provide, in a single chromatography step and good yield, pure Taq Pol suitable for molecular biology applications. Materials and methods MaterialsThe pQE-70 vector and E. coli M15 (pREP4) strain was purchased from Qiagen (Germany). E. coli XL-1 Blue strain was purchased from Stratagene (USA). Taq Pol, Pfu Pol and deoxynucleotide triphosphates (dNTPs) were purchased from Promega (UK). The pTacTaq vector, carrying the Taq Pol coding region was a kind gift of Dr. J. F. Davidson (Department of Pathology, University of Washington, Seattle, USA). Protamine sulfate was obtained from Sigma-Aldrich (USA). Construction of the ligand libraryThe rational design and the chemical synthesis of the ligand library were...

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“…The design of these dyes is performed on the basis of either complementarity by studying a natural ligand-protein complex or mimicking a natural biological recognition interaction [14,15].…”

Section: Introductionmentioning

confidence: 99%

Assessment of Pseudoaffinity Chromatography Using Textile Dyes for Isolation of Buffalo Pituitary Luteinizing Hormone

Arora

Patel

Muralidhar

2012

ISRN Chromatography

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Extensive investigation has been carried out to elucidate the mechanisms involved in pseudoligand affinity chromatography using textile dyes, and, empirically, it has been attributed to the chemical and steric structures of dye and protein. Possibly, a variety of interactions especially ionic and/or hydrophobic influence with a varying share in the binding and differ from protein to protein and from dye to dye. In this study, we have attempted to understand the effect of various biophysical parameters like the nature of the eluant, pH, and ionic strength on the binding of crude luteinizing hormone (LH) with various triazine-based dyes and thus predict their nature. Based on the elution patterns, cibacron and reactive brown suggested a dual electrostatic and hydrophobic nature. Reactive blue and reactive yellow reflected a major electrostatic/ionic nature with yellow offering 50-fold purification in a single step, while reactive red and reactive green had a predominant hydrophobic nature. Appreciably, reactive red was binding LH very tightly unlike other dyes, and addition of the arginine in the elution buffer substantially weakened the protein-dye interactions. pH was observed to be a principal factor assisting the protein-dye binding as well as hydrophobicity of the dye and the proteins.

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Affinity chromatography matures as bioinformatic and combinatorial tools develop

Cited by 128 publications

References 163 publications

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

Assessment of Pseudoaffinity Chromatography Using Textile Dyes for Isolation of Buffalo Pituitary Luteinizing Hormone

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