Polyelectrolytes and Polycomplexes in Biotechnology

Detta är en avhandling från Department of Biotechnology, Center for Chemistry and Chemical Engineering, P.O. Box 124, S-221 00 Lund, Sweden

Sammanfattning: Polyelectrolytes and polyelectrolyte complexes (PECs) are characterized by many useful properties that endow them with high potential in different areas of biotechnology. This thesis describes the development and analyses of some new systems and methods utilizing poly(acrylic acid) (PAA), poly(methacrylic acid) (PMA), poly (N-ethyl-4-vinylpyridinium bromide) (PEVP) and PMA-PEVP complexes. The model of chaperone action when the misfolded inactive forms of polypetides are removed from the medium preventing their aggregation was developed using PEVP and conjugates of PMA with monoclonal antibodies (IgG) specific to inactivated glyceraldehyde-3-phosphate dehydrogenase (iGAPDH). PMA-IgG conjugates “recognized” and bound iGAPDH in the solution of inactivated enzyme. Addition of stoichiometric amount of PEVP resulted in quantitative precipitation of the formed complex PMA-IgG-iGAPDH-PEVP which was removed by centrifugation. As the next step in the model development iGAPDH was covalently coupled to PMA. pH-dependent properties of IgG- and iGAPDH-PMA conjugates have been studied using turbidimetry and light scattering. The immunocomplexes iGAPDH / IgG-PMA and iGAPDH-PMA / IgG-PMA were shown to precipitate in acidic media. The competition of free iGAPDH and iGAPDH-PMA for binding with IgG-PMA and the dynamic release of refolded GAPDH, which looses affinity to IgG-PMA, into solution could be used for simulating chaperone action. Nonstoichiometric PEC formed by PMA and PEVP udergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5-6.5 depending on the ionic strength and PEVP/PMA ratio in the complex. Antigen, iGAPDH, was covalently coupled to PEVP and the resulting conjugate was used for affinity precipitation of IgG using nonstoichiometric PEC. On the base of the developed affinity precipitation procedure a new method of the production of monovalent Fab fragments of IgG has been developed. Proteolysis of IgG in the presence of iGAPDH-PEVP conjugate followed by i) precipitation induced by PMA addition and pH shift from 7.3 to 6.5 and ii) elution at pH 4.0 resulted in 90 % immunologically competent Fab fragments. PAA was electrostatically attached to the surface of STREAMLINE DEAE beads in order to prevent binding of cells to the resin in expanded bed chromatography. The modified matrix was resistant towards interactions with the biomass and was successfully used for isolation of BSA from the model mixture containing bovine serum albumin, lysozyme (positively charged at applied conditions) and yeast cells. A new technique for coating strong anion exchangers with PAA layer has been developed. A layer of PAA was attached to the surface of Amberlite beads via electrostatic interactions followed by a covalent cross-linking of PAA. The cross-linked-PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth. Native Amberlite could not be used due to the immediate clogging of the column with the biomass and the collapse of the expanded bed.

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