Construction of a vector permitting immobilization and visualization of enzymes on compatible polymeric surfaces

The general application of immobilized proteins and enzymes has played a central role in the expansion of biotechnology and synthesis-related industries. As the efficiency, selectivity and maximum attainable loading of enzymes upon commercially produced surfaces bear strongly on product performance, much effort has been invested to introduce user-friendly, selective and vectorially orienting methods of immobilization for any protein. Continuing along this theme, we developed a two-component system described by an expression vector and an activated polymeric surface. The expression vector pETM-11 was chosen for the reason that its histidine-rich tag could facilitate immobilization of expressed proteins and direct their surface orientation. The strategy adopted was to fuse the vector with a GFP (green fluorescent protein) gene followed by a multiple cloning site. In principle, this multiple cloning site can be used to introduce the gene of any protein, barring size constraints, affording ready-to-immobilize fusion proteins with native-like function in near-native surroundings. It would follow that the location and loading of immobilized proteins can be conveniently assessed on the basis of fluorescence emitted by the accompanying GFP. Since the distance separating protein and surface is large in comparison to many immobilization methods, immobilized enzymes are expected to display native-like characteristics, which may prove advantageous in certain cases. The immobilization strategy also requires that a compatible substrate surface would respond selectively to the his-tag. To this end, several surfaces bearing linkers that could interact covalently or electrostatically with the imidazole functional group were prepared. Current work is directed at optimizing the conditions of immobilization, quantifying the bound protein, and characterizing its function. We are also exploring secondary applications that might exploit inherent physico-chemical properties of this system. For example, cleaving the adapter portion enzymatically or altering the electrostatic interaction between surface and protein could be used as a selective method of purification, with the release of protein monitored visually. Construction of a vector permitting immobilization and visualization of enzymes on compatible polymeric surfaces