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Controlling the orientation of immobilized proteins by genetic circular permutation

Publication ,  Journal Article
Piervincenzi, RT; Chilkoti, A
Published in: Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
December 1, 1999

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 a carboxylic acid-functionalized surface by tendamistat's N-terminal amine. Protein immobilization by native N or C-terminus, 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.

Duke Scholars

Published In

Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings

ISSN

0589-1019

Publication Date

December 1, 1999

Volume

1

Start / End Page

93
 

Citation

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ICMJE
MLA
NLM
Piervincenzi, R. T., & Chilkoti, A. (1999). Controlling the orientation of immobilized proteins by genetic circular permutation. Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings, 1, 93.
Piervincenzi, R. T., and A. Chilkoti. “Controlling the orientation of immobilized proteins by genetic circular permutation.” Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 1 (December 1, 1999): 93.
Piervincenzi RT, Chilkoti A. Controlling the orientation of immobilized proteins by genetic circular permutation. Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings. 1999 Dec 1;1:93.
Piervincenzi, R. T., and A. Chilkoti. “Controlling the orientation of immobilized proteins by genetic circular permutation.” Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings, vol. 1, Dec. 1999, p. 93.
Piervincenzi RT, Chilkoti A. Controlling the orientation of immobilized proteins by genetic circular permutation. Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings. 1999 Dec 1;1:93.

Published In

Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings

ISSN

0589-1019

Publication Date

December 1, 1999

Volume

1

Start / End Page

93