- Piervincenzi, R.T. and Chilkoti, A., Controlling the orientation of immobilized proteins by genetic circular permutation,
Proceedings of the First Joint BMES/EMBS Conference. 1999 IEEE Engineering in Medicine and Biology 21st Annual Conference and the 1999 Annual Fall Meeting of the Biomedical Engineering Society (Cat. No.99CH37015), vol. vol.1
pp. 93 vol.1 - [IEMBS.1999.802129] .
(last updated on 2007/04/12)
Controlling the orientation of immobilized proteins is critical to their surface activity. However, most currently used protein immobilization strategies result in a wide range of surface orientations. Because termini are ubiquitous to proteins, we have investigated their use as unique sites for immobilization to create homogeneously oriented protein surfaces with optimized activity. We demonstrate proof-of-concept, for a model protein tendamistat, by immobilizing it on a surface specifically by its N-terminal amine by: expression as a C-terminal fusion to thioredoxin; blocking accessible lysine residues; liberation of the N-terminal amine by proteolytic cleavage of the fusion protein; and immobilization to carboxylic acid functionalized surface by tendamistat's N-terminal amine. Protein immobilization by native N or C-termini is, however, of limited utility because the orientation specified by the location of the native termini may be inappropriate for a target application. This limitation can be circumvented by using genetic circular permutation to reposition the termini on the protein scaffold. In studies to date, we have shown that controlling the location of the N-terminus of tendamistat by circular permutation, and consequently the site of immobilization, has a dramatic effect on its surface activity. We are currently extending this approach to incorporate genetically-encoded C-terminal peptide tags in permuted proteins, and complementarily, by exploiting enzyme-assisted reverse proteolysis to immobilize permuted proteins by their engineered C-termini
binding energy;biochemistry;biological specimen preparation;catalysis;genetics;molecular biophysics;molecular orientation;proteins;surface energy;