We propose to develop a new system for site specific labeling proteins in live cells based on the bacterial transpeptidase sortase A (SrtA). Using a combination of protein engineering and directed evolution, we will develop SrtA variants capable of efficiently functioning within mammalian cells. We will also develop exceptionally small (<2KDa), brightly fluorescent membrane permeable labels as well as substrates which will fluoresce only upon attachment to the target protein. The reduced size of these labels coupled with their strong fluorescence will permit monitoring of proteins at lower, more physiological expression levels while reducing any potential for steric interference. Initial work will focus on labeling secretory proteins, but the technology will be applicable to almost any protein located in a number of cellular compartments.

Public Health Relevance

We will develop a novel enzymatic method for labeling specific proteins with small fluorescent molecules in live cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087985-05
Application #
8539803
Study Section
Special Emphasis Panel (ZRG1-BST-K (50))
Program Officer
Deatherage, James F
Project Start
2009-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2013
Total Cost
$298,304
Indirect Cost
$118,603
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461