Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

<p>Abstract</p> <p>Background</p> <p>Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library.</p> <p>Results</p> <p>Both phages and <it>E. coli </it>cells pass non-hindered through the interconnected pores of macroporous gel, so called <it>cryogel</it>. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. <it>E. coli </it>cells were applied on the column for infection with the specifically bound phages.</p> <p>Conclusion</p> <p>Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection.</p

Stimulus responsive surfaces. Possible implications for bioseparation, cell cultivation and drug delivery.

Abstract is not availabl

Affinity processing of cell-containing feeds using monolithic macroporous hydrogels, cryogels.

Monolithic macroporous hydrogels, "cryogels," are produced by polymerization in a partially frozen state when the ice crystals perform as a porogen. Cryogels have a unique combination of properties: (i) large (10-100 microm) pores; (ii) minimal non-specific interactions due to the hydrophilic nature of the polymers; (iii) porosities exceeding 80-90%; (iv) good mechanical stability. These properties of cryogels allow for their application for direct capture of extracellularly expressed histidine-tagged protein from the fermentation broth and separation of different cell types

Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption

Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed, only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which other-wise bound,strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles

Graft polymerization of vinyl monomers inside macroporous polyacrylamide gel, cryogel, in aqueous and aqueous-organic media initiated by diperiodatocuprate(III) complexes

Graft polymerization initiated by diperiodatocuprate(III) complex (Cu(III)) initiator was found to be an effective and convenient method for graft polymerization of vinyl monomers onto macroporous polyacrylamide gels, the so-called cryogels (pAAm-cryogels). The effect of time, temperature, monomer and initiator concentration during the graft polymerization in aqueous and aqueous-organic media was studied. The graft polymerization of water-soluble monomers as [2-(methacryloyloxy)ethyl]-tri-methylammonium chloride, 2-hydroxyethyl methaerylate, N-isopropylacrylamide, and N,N-dimethylacrylamide proceeds with higher grafting yield in aqueous medium, as compared with that in aqueous-organic media. Graft polymerization in aqueous-organic media such as water-DMSO solutions allows grafting of water-insoluble monomers such as glycidyl methacrylate and N-tert-butylacrylamide with high grafting degrees of 100 and 410%, respectively. It was found that the deposition of initiator on the pore surface of cryogels promoted graft polymerization by facilitating the formation of the redox couple Cu(III)-acrylamide group. (c) 2006 Wiley Periodicals, Inc

Precipitation of proteins: Nonspecific and specific.

Abstract is not availabl

Anion-exchange supermacroporous monolithic matrices with grafted polymer brushes of N,N-dimethylaminoethyl-methacrylate

Graft polymerization using potassium diperiodatocuprate as initiator was found to be an effective and convenient method for grafting functional polymer of N,N-dimethylaminoethyl methacrylate (DMAEMA) onto superporous polyacrylamide gels, so-called cryogels (pAAm cryogels). It was possible to achieve grafting degrees up to 110% (w/w). The two-step graft polymerization i.e. first activation of the matrix followed by displacement of initiator solution with the monomer solution, decreased pronouncedly the soluble homopolymer formation. The efficiency of graft polymerization using a two-step technique increased up to 50% (w/w) at a monomer conversion of 10%, compared to 10% graft efficiency with 60-70% monomer conversion for one-step direct graft polymerization. The pAAm cryogels grafted in one-step and two-step procedures, respectively, behaved similarly when binding low-molecular weight ligand but showed very different behavior for sorption of a high-molecular-weight ligand, bovine serum albumin (BSA). The differences in behavior were rationalized assuming different structure of the graft polymer layers and tentacle-type BSA binding to the grafted polymer. (c) 2005 Elsevier B.V. All rights reserved

Binding of adenosine to pendant phenylboronate groups of thermoresponsive copolymer: a quantitative study

Binding of adenosine to the thermosensitive copolymer of N-isopropylacrylamide and 3-(acrylamido)aminophenylboronic acid (82:18, m ($) over bar (n)=47000 g center dot mol(-1)) was studied by equilibrium dialysis at 22 degrees C and 37 degrees C, in a 0.1 m glycine buffer containing 0.1 m NaCl at pH 9.2. The copolymer exhibited a the phase transition temperature (T-p) of 26.5 degrees C under the above conditions. At 22 degrees C the binding of adenosine to the water-soluble copolymer was well described by a Langmuir model, accounting for preferential ionisation of the boronate-nucleoside complexes and, therefore, restricted reactivity of the rest of boronates. At saturation, the copolymer contained 38% of its phenylboronic acid groups in the form of complexes, whereas the association constant was 1400 m(-1). At 37 degrees C no binding of adenosine to thermally precipitated copolymer was found, presumably owing to interaction of the phenylboronates with hydrophobic segments of polyNIPAM. At high loading of the copolymer by the reversibly bound adenosine the T (p) steeply increases with increasing fraction of the phenylboronate-adenosine complexes in the chains. The increase of the T-p observed above the saturating adenosine concentration (> 1 x 10(-3) m, 22 degrees C) very probably testifies to competition of the nucleoside with hydrophobic polyNIPAM segments for binding to the pendant phenylboronates

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications

Macroporous gels (MGs) with a broad variety of morphologies are prepared using the cryotropic gelation technique, i.e. gelation at subzero temperatures. These highly elastic hydrophilic materials can be produced from practically any gel-forming system with a broad range of porosity extending from elastic and porous gels with pore sizes up to 1.0 mu m to elastic and sponge-like gels with pore sizes up to 100 mu m. The versatility of the cryogelation technique is demonstrated by use of different chemical reactions (hydrogen bond formation, chemical cross-linking of polymers, free radical polymerization) mainly in an aqueous medium. Appropriate control over solvent crystallization (formation of solvent crystals) and rate of chemical reaction during the cryogelation allows the reproducible preparation of cryogels with tailored properties. Different approaches, such as chemical modification of reactive groups, grafting of the pore surface with an appropriate polymer, or direct copolymerization with functional monomers are used for control of the surface chemistry of MGs. Typically, MGs with pore sizes up to 1.0 mu m are produced in the shape of beads and MGs with pore size up to 100 mu m are prepared as monoliths, discs, and sheets. The difference in porous structure of MGs defines the main applications of these porous materials. Elastic beaded MGs are mostly used as carriers for. cell and enzyme immobilization or for capture of low-molecular weight targets from particulate-containing fluids in expanded-bed mode. However, the elastic and sponge-like MG monoliths with interconnected pores measuring hundreds of gm have been successfully used as monolithic columns for chromatography of particulate-containing fluids (crude cell homogenates, viruses, whole cells, wastewater effluents) and as three-dimensional scaffolds for mammalian cell culture applications