Use of the avidin (imino)biotin system as a general approach to affinity precipitation

Garret‐Flaudy, Frédéric; Freitag, Ruth

doi:10.1002/1097-0290(2000)71:3<223::aid-bit1012>3.3.co;2-l

Cited by 7 publications

(12 citation statements)

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“…This unusual property of PNIPAAm and its copolymers facilitated its use for the development of innovative methods for the delivery of drugs, peptides, and proteins 6, 7. PNIPAAm has been developed for use in a number of fields including bioseparation, chemical/pH sensors, actuators, and chemical valves in microfluidics 8–13…”

Section: Introductionmentioning

confidence: 99%

“…6,7 PNI-PAAm has been developed for use in a number of fields including bioseparation, chemical/pH sensors, actuators, and chemical valves in microfluidics. [8][9][10][11][12][13] Modifying surfaces with PNIPAAm allows the surface to demonstrate temperature responsive wettability changes in aqueous environments. PNIPAAm modification can be done through a variety of methods.…”

Section: Introductionmentioning

confidence: 99%

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Cell growth and detachment from protein‐coated PNIPAAm‐based copolymers

Moran

Carroll

Селезнева

et al. 2007

J Biomedical Materials Res

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The cultivation of cells requires the use of unfavorable proteolytic enzymes, which cause cell-surface modification and also need considerable optimization. Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without using enzymes by utilizing the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). Copolymers of PNIPAAm and N-tert-butyl-acrylamide (NtBAm) with varying ratios were synthesized and solvent cast. The copolymer films are coated with cell adhesion promoters such as collagen, poly-L-lysine, and laminin to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue and PicoGreen assays is similar for coated copolymer films and standard tissue culture plastic controls. Deposition of cell adhesion promoters onto the copolymer films was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell detachment from the copolymer films is dependent on copolymer composition and is not affected by the surface coatings of extracellular matrix (ECM) proteins. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cell growth and detachment from protein‐coated PNIPAAm‐based copolymers

Moran

Carroll

Селезнева

et al. 2007

J Biomedical Materials Res

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“…In our experience, the heterogeneous precipitation behavior of many of the early AML also caused some problems with the recovery/quantitative removal of the AML [9,10]. Since then, a second-generation type of AML has been developed in our group, which overcomes many of these problems [9,11,12]. The base material of this AML is poly(N-isopropylacrylamide), polyNIPAM, a well-known temperature-responsive polymer [13] with a CST in pure water of 32°C.…”

Section: Technical Reportmentioning

confidence: 99%

“…Biotin-AML Avidin [11,19] Protein A-AML Antibodies [14] (triple helix motif) ssDNA-AML (e.g. (CTT) 7 -polyNIPAM) (Plasmid) DNA (triple helix interaction) [16,20] polyT-AML mRNA [15] Fusion proteins (peptide tag) Anti FLAG-tag antibody-AML FLAG-tag Anti APP-tag antibody-AML APP-tag [14] Ni-NTA-AML His 6 -tag [14] Avidin-AML a)…”

Section: Purification Of Referencementioning

confidence: 99%

Affinity precipitation an option for early capture in bioprocessing

Freitag

Schumacher²,

Hilbrig³

2007

Biotechnology Journal

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Affinity precipitation is a bioseparation technique where the affinity ligand is coupled to a stimuliresponsive polymer. Stimuli-responsive polymers show abrupt, yet reversible, phase transition (precipitation) in response to a small change in an environmental parameter. The corresponding ligand conjugates can be used to co-precipitate and thereby capture and isolate target molecules from complex solutions such as culture supernatants and cell lysates. The approach is compatible with a 'discardibles only' type of downstream process and can be scaled over several orders of magnitude. This report discusses the set-up and development of affinity precipitation procedures, the related instrumentation and scale up, as well as applications for the isolation of proteins and polynucleotides.

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“…11 PNIPAM coupled to an iminobiotin affinity ligand was used to isolate lysozyme from fetal calf sera. 12 In connection with our interest in coupling natural products to their cellular receptors, 13 we considered the attachment of natural products to PNIPAM polymers to facilitate characterization of the immobilized natural product. 14 Affinity chromatography is based on the principle of specific protein-ligand interactions and is a common tool for purification of proteins, antibodies, and receptor-ligand complexes.…”

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confidence: 99%

Synthesis, Characterization, and Utility of Thermoresponsive Natural/Unnatural Product Macroligands for Affinity Chromatography

et al. 2006

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The synthesis and characterization of thermoresponsive, water-soluble poly-N -isopropyl acrylamide (PNIPAM) derived macroligands displaying cyclosporin A (CsA) and dexamethasone (Dex) for use as novel affinity resins is described. Characterization of these soluble macroligands, including ligand loading and integrity was determined by 1 H NMR spectroscopy. One of the CsA-macroligands was used in a protein affinity experiment to capture known binding proteins of CsA, the cyclophilins, from Jurkat T-cell lysates.Organisms utilize biopolymers in the form of DNA for information storage and in the form of proteins for cellular structure and machinery. Most functional biopolymers respond to external stimuli such as heat and pH in an all-or-nothing, or at least a highly non-linear mode. 1 Most common among such biopolymers are proteins that precipitate or denature on heating. Recently, progress has been made toward the synthesis of synthetic functional polymers that mimic biopolymers by responding in a desired way to external environmental stimuli such as temperature, pH, and either electric or magnetic fields. 2 These highly nonlinear responses of synthetic polymers occur primarily in water, but also in organic solvents 3 or polymer blends. 4 When this stimulus-responsive behavior occurs in aqueous solution, these polymers have potential as useful tools for biotechnological and medicinal research. Among the synthetic stimulus-responsive polymers, temperature responsive polymers have been the most extensively studied. 5 This kind of polymer is soluble below its lower critical solution romo@mail.chem.tamu.edu. Supporting Information Available: General experimental procedures and characterization data for compounds 3a-b and 7−9. Representative 1 H NMR spectra for macroligands 4a-4d and 10a-e and procedure for the affinity experiment. Copolymers of poly(N-isopropylacrylamide) (PNIPAM) and derived functionalized polymers have been shown to be thermoresponsive, exhibiting quantitative inverse temperaturedependent solubility in water. 8,9 These and other thermo-responsive polymers have enabled the design of smart chemical catalysts having activities that can be switched off and on by precipitation and redissolution based on reaction temperature. 8,9 In addition, these polymers are useful in syntheses using biological catalysts. 10 Most pertinent to the described studies herein are previous uses of PNIPAM-derived thermoresponsive polymers to purify known proteins. An imidazole-PNIPAM thermoresponsive polymer loaded with Cu(II) ions was used for the affinity purification of proteins from cereals ( Figure 1). 11 PNIPAM coupled to an iminobiotin affinity ligand was used to isolate lysozyme from fetal calf sera. 12 In connection with our interest in coupling natural products to their cellular receptors, 13 we considered the attachment of natural products to PNIPAM polymers to facilitate characterization of the immobilized natural product. 14 Affinity chromatography is based on the principle of specific protein-ligand inter...

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Use of the avidin (imino)biotin system as a general approach to affinity precipitation

Cited by 7 publications

References 0 publications

Cell growth and detachment from protein‐coated PNIPAAm‐based copolymers

Cell growth and detachment from protein‐coated PNIPAAm‐based copolymers

Affinity precipitation an option for early capture in bioprocessing

Synthesis, Characterization, and Utility of Thermoresponsive Natural/Unnatural Product Macroligands for Affinity Chromatography

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