Protein switches hold great promise as selective protein therapeutics and as biosensors for diagnostic and basic science applications;however, the creation of such proteins has proven difficult. We propose to create protein switches for these applications using a combinatorial method we developed that views all existing proteins as potential input and output modules for the desired switch. We will create a protein switch that will activate a prodrug only in cancer cells. Such a switch would have potential as a targeted therapeutic for the treatment of cancer. In addition, we will create switches to be used as sensors for kinases in vitro and in live cells. Switches to be used as sensors will be developed on a platform such that sensors for other proteins will be more readily created. Experiments proposed will also inform the study of how best to create switches

Public Health Relevance

: Engineered protein switches hold great promise as selective protein therapeutics and as biosensors for diagnostic and basic science applications. We will develop switches designed for the treatment of cancer and switches designed to be used as sensors for the detection and quantification of important protein kinases in vitro and in live cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM066972-10S1
Application #
8703351
Study Section
Program Officer
Gerratana, Barbara
Project Start
2003-07-01
Project End
2017-02-28
Budget Start
2013-04-01
Budget End
2014-02-28
Support Year
10
Fiscal Year
2013
Total Cost
$61,244
Indirect Cost
$19,980
Name
Johns Hopkins University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc et al. (2014) The High-Affinity Maltose Switch MBP317-347 has Low Affinity for Glucose: Implications for Targeting Tumors with Metabolically Directed Enzyme Prodrug Therapy. Chem Biol Drug Des 83:266-71
Chaikind, Brian; Ostermeier, Marc (2014) Directed evolution of improved zinc finger methyltransferases. PLoS One 9:e96931
Kanwar, Manu; Wright, R Clay; Date, Amol et al. (2013) Protein switch engineering by domain insertion. Methods Enzymol 523:369-88
Guntas, Gurkan; Kanwar, Manu; Ostermeier, Marc (2012) Circular permutation in the ýý-loop of TEM-1 ýý-lactamase results in improved activity and altered substrate specificity. PLoS One 7:e35998
Cheung, Luthur Siu-Lun; Kanwar, Manu; Ostermeier, Marc et al. (2012) A hot-spot motif characterizes the interface between a designed ankyrin-repeat protein and its target ligand. Biophys J 102:407-16
Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew et al. (2011) Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA. Biotechnol Bioeng 108:2535-43
Wright, Chapman M; Wright, R Clay; Eshleman, James R et al. (2011) A protein therapeutic modality founded on molecular regulation. Proc Natl Acad Sci U S A 108:16206-11
Heins, Richard A; Choi, Jay H; Sohka, Takayuki et al. (2011) In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells. PLoS One 6:e27302
Ostermeier, Marc (2009) Designing switchable enzymes. Curr Opin Struct Biol 19:442-8
Berrondo, Monica; Ostermeier, Marc; Gray, Jeffrey J (2008) Structure prediction of domain insertion proteins from structures of individual domains. Structure 16:513-27