Affinity purification of natural ligands.

Immobilization of proteins, nucleic acids, and other bioligands is not always straightforward since they are often large molecules with numerous chemically reactive groups that can all participate in the immobilization process through physical adsorption, ionic binding, or covalent linkage. Protocols for some of the most frequently used matrix-activation systems are described in this unit. For agarose, protocols are given for cyanogen bromide, p-nitrophenyl chloroformate, tresyl chloride, and cyanuric chloride. Tosyl chloride is used to activate cellulose, and cyanuric chloride is also used to activate aminopropyl silica gel. Activation of magnetic beads with cyanogen bromide is described, and a protocol is provided for reacting the aldehyde groups of glyoxal agarose beads with the primary amine groups of ligands, with subsequent reduction of the formed Schiff base to yield a stable matrix-ligand bond.

Can tobacco have a potentially beneficial effect to our health?

With urgent pressure to clean up the contaminated environment, new approaches are needed. Phyto- and rhizoremediation using plants and related bacteria is a promising approach, but has its inborn limitations. To overcome the slow performance of the process, transgenic plants have been prepared specifically tailored for phytoremediation purposes. Our projects addressed a group of widespread synthetic organic xenobiotics, polychlorinated biphenyls (PCBs), and heavy metals as representatives of inorganic contaminants. Beside basic research studies in the field of phyto/rhizoremediation of the mentioned toxicants we focused on genetically modified plants as a highly promising tool for these purposes. We tried to prepare tobacco plants expressing the bacterial enzyme responsible for cleaving PCBs, coded by the gene bphC from the bacterial biphenyl operon. The expression of bphC product in fusion with the green fluorescent protein is described together with evaluation of the twice increased resistance of transgenic seeds towards PCBs. The other model is addressing improvement of cadmium accumulation by preparing plants bearing fused transgenes of metal binding protein (yeast metallothionein) with an introduced additional metal binding domain--polyhistidine anchor with high affinity to metals. The genetically modified plants exhibit 190% Cd accumulation of the control in harvestable parts, higher resistance and lower Cd content in roots. The performance of the plants in real contaminated soil is also evaluated.