Alexander E. Ivanov

Conjugation of Penicillin Acylase with the Reactive Copolymer of N‐Isopropylacrylamide: A Step Toward a Thermosensitive Industrial Biocatalyst

Conjugation of penicillin acylase (PA) to poly‐N‐isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10‐fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4‐fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 106 and, presumably, cross‐linked protein‐polymer aggregates of MW > 107. Michaelis constant for 5‐nitro‐3‐phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 μM) was found to be slightly higher than that of the free enzyme (12 μM), and evaluation of Vmax testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM‐cross‐linked PA retained its capacity to synthesize cephalexin from d‐phenylglycin amide and 7‐aminodeacetoxycephalosporanic acid. The synthesis‐hydrolysis ratios of free and polyNIPAM‐cross‐linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of β‐lactam antibiotics, can be successfully eliminated by cross‐linking penicillin acylase to a smart polymer (i.e., polyNIPAM).

Metal-chelate affinity precipitation of proteins using responsive polymers

Affinity precipitation of proteins uses polymers capable of reversible soluble-insoluble transitions in response to small environmental changes (temperature, pH or solvent composition). Here we describe protocols for (i) the synthesis of responsive polymers with specific affinity to target proteins and (ii) the purification of proteins using these polymers. The purification is based on precipitation of the affinity complex between the protein and the polymer, which is induced by environmental changes. This separation strategy is simpler and more cost effective than conventional affinity column chromatography. Specifically, we describe the synthesis of thermoresponsive 1-vinylimidazole:N-isopropylacrylamide copolymers. The whole procedure takes 2–3 h when applied to purification of recombinant His-tag proteins or proteins with natural metal binding groups by means of metal chelate affinity precipitation. Optimization of the polymer composition and the type of chelating ions allows for target protein yields of 80% and higher.

Boronate-containing polymer brushes: Characterization, interaction with saccharides and mammalian cancer cells

Boronate-containing polymer brushes were synthesized by free radical copolymerization of N,N-dimethylacrylamide (DMAA) and N-acryloyl-m-phenylboronic acid (NAAPBA) (9:1) on the surface of 3-mercaptopropyl-silylated glass plates and capillaries. The brushes were characterized with time-of-flight secondary ion mass-spectrometry (ToF SIMS), atomic force microscopy and contact angle measurements. Fructose caused a well-expressed drop spreading on the surface of copolymer-grafted glass, due to the strong interaction with the boronate groups. Sedimentation of murine hybridoma cells M2139 or human myeloid leukemia cells KG1 onto the DMAA-NAAPBA copolymer-grafted glass plates from 10 mM phosphate buffer solution (pH 8.0) resulted in the cell adhesion. The adhered M2139 and KG1 cells could be quantitatively detached from the grafted plates with 0.1 M fructose, which competed with cell surface carbohydrates for binding to the boronates. Evaluation of the binding strength between M2139 cells and the copolymer brush was performed by exposure of the adhered cells to a shear stress. Detachment of a fraction of 18% of the adhered M2139 cells was obtained at a shear force of 1400-2800 pN/cell generated by the running phosphate buffer (pH 8.0), whereas the remaining adhered cells (70%) could be detached with 0.1 M fructose dissolved in the same buffer. Possible applications of the boronate-containing polymer brushes to affinity cell separation can be based upon the facile recovery of the attached cells.

Photosensitive copolymer of N-isopropylacrylamide and methacryloyl derivative of spyrobenzopyran

The copolymer of N-isopropylacrylamide (NIPAM) and methacryloyl derivative of spirobenzopyran (MSBP) with a molecular weight of 21 000 g/mol and the average molar MSBP content of 1.9% was prepared by free radical polymerization. The copolymer displayed its phase transition in water in the temperature range of 30-50 degreesC. UV irradiation of its aqueous solution caused photoinduced transformation of MSBP units into their coloured merocyanine forms, while the cloud point of the irradiated copolymer shifted by ca. 10 degreesC to lower temperatures. During a long-term exposure to daylight (20 days) the copolymer gradually elapsed to its colourless spiropyran form, the process being ca. 100-times slower than that for monomeric MSBP. Due to the slow reverse isomerization of its merocyanine form and low solubility in water at room temperature the UV irradiated copolymer could be quantitatively separated from aqueous solution by centrifugation

Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography.

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).

Boronate-containing polymers form affinity complexes with mucin and enable tight and reversible occlusion of mucosal lumen by poly (vinyl alcohol) gel

Copolymers of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) formed insoluble interpolymer complexes with mucin from porcine stomach at pH 9.0. The complex formation based on boronate-sugar interactions took place between the similarly charged macromolecules and resulted in coacervate particles formation, which depended both on pH and ionic strength of the solution. The coacervation rate displayed a maximum at the intermediate DMAA-NAAPBA copolymer: mucin weight ratio, that is a pattern typical of interpolymer complex formation. The effective hydrodynamic particle diameter of the coacervates monotonously grew from 155+/-20 nm up to 730+/-120 nm in 2 days in 0.1M sodium bicarbonate buffer solution, pH 9.0. Electrophoretic mobility of the resultant nanoparticles was intermediate between those of individual polymers, whereas the particles zeta-potential was -7.5+/-0.4 mV in the above buffer solution. Pre-treatment of the inner mucosal epithelium of excised male pig urethras with 5% (w/v) solutions of acrylamide-NAAPBA or DMAA-NAAPBA copolymers at pH 8.8 allowed for tight occlusion of the lumen by poly(vinyl alcohol)-borax gel injected via a two-way catheter. Leakage of 0.15M NaCl solution through the thus occluded organs could be prevented, while the leakage through the organs occluded by the gel without the pre-treatment was unavoidable. The gel plug could be quickly dissolved on demand after injection of 5% (w/v) aqueous fructose solution into the lumen. The described technique may be useful for temporal occlusion of mucosal lumens in living organisms. In contrast to the conventional mucoadhesive polymers like polyacrylic acid or chitosan, the boronate-containing copolymers display their mucoadhesivity at weakly alkaline pH of 8-9 and physiological ionic strength.

Effect of temperature upon the chromatography of proteins on porous glass, chemically coated with N-isopropylacrylamide copolymer

Abstract Wide-pore glass chemically coated with a copolymer of N-isopropylacrylamide (NIPAA) and N-hydroxyethylacrylamide (70:30) was studied as a weak-hydrophobic sorbent for chromatography of proteins. The temperature dependence of the lysozyme chromatographic retention points to the maximum near a lower critical solution temperature of the copolymer (LCST, 41°C). Nevertheless, log k′ vs. [(NH4)2SO4] plots found for lysozyme at 25°C and 45°C are only slightly different and indicate almost zero free energies of interaction between the protein and the copolymer in 0.01 M potassium phosphate solution, pH 7.5. No temperature-modulated desorption of immunoglobulin G adsorbed to the copolymer-coated glass at 45°C was observed when cooling the column to 30°C. Changes in protein interactions with the polymer grafts are apparently too weak to ensure an effective control of protein adsorption with temperature shift near LCST.

Evaluation of boronate-containing polymer brushes and gels as substrates for carbohydrate-mediated adhesion and cultivation of animal cells

Boronate-containing thin polyacrylamide gels (B-Gel), polymer brushes (B-Brush) and chemisorbed organosilane layers (B-COSL) were prepared on the surface of glass slides and studied as substrates for carbohydrate-mediated cell adhesion. B-COSL- and B-Brush-modified glass samples exhibited multiple submicron structures densely and irregularly distributed on the glass surface, as found by scanning electron microscopy and atomic force microscopy. B-Gel was ca. 0.1 mm thick and contained pores with effective size of 1-2 microm in the middle and of 5-20 microm on the edges of the gel sample as found by confocal laser scanning microscopy. Evidence for the presence of phenylboronic acid in the samples was given by time-of-flight secondary ion mass-spectrometry (ToF SIMS), contact angle measurements performed in the presence of fructose, and staining with Alizarin Red S dye capable of formation specific, fluorescent complexes with boronic acids. A comparative study of adhesion and cultivation of animal cells on the above substrates was carried out using murine hybridoma M2139 cell line as a model. M2139 cells adhered to the substrates in the culture medium without glucose or sodium pyruvate at pH 8.0, and then were cultivated in the same medium at pH 7.2 for 4 days. It was found that the substrates of B-Brush type were superior both regarding cell adhesion and viability of the adhered cells, among the substrates studied. MTT assay confirmed proliferation of M2139 cells on B-Brush substrates. Some cell adhesion was also registered in the macropores of B-Gel substrate. The effects of surface microstructure of the boronate-containing polymers on cell adhesion are discussed. Transparent glass substrates grafted with boronate-containing copolymers offer good prospects for cell adhesion studies and development of cell-based assays.

Polymer versus monomer as displacer in immobilized metal affinity chromatography.

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.

Reversible Conformational Transitions of a Polymer Brush Containing Boronic Acid and its Interaction with Mucin Glycoprotein.

Reversible changes of the height of a polymer brush containing phenylboronic acid were studied. The polymer brush thickness underwent reversible changes of 0.5-1 nm, in response to the changes in composition of the contacting aqueous phase from deionized water to bicarbonate buffer and vice versa, apparently due to the conformational transition of the weak polyelectrolyte to the more extended electrically charged state. Adsorption of mucin glycoprotein to the polymer brush took place due to boronate/sugar interactions between the glycoprotein and the graft copolymer and resulted in further increase of the brush height by ca. 1.5 nm, as observed by means of spectral correlation spectroscopy and ellipsometry.