Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells.
We demonstrate the evolution of the PylRS/tRNA(CUA) pair for genetically encoding photocaged cysteine. By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, we establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. We demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.
Medical Research Council Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
Duy P
DP
Nguyen
Mohan
M
Mahesh
Simon J
SJ
Elsässer
Susan M
SM
Hancock
Chayasith
C
Uttamapinant
Jason W
JW
Chin
D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D019143:Evolution, Molecular; D031541:Fluorescence Resonance Energy Transfer; D005815:Genetic Code; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure
D010450:Endopeptidases; C099488:TEV protease; D003545:Cysteine