WLODARCZYK JAKUB (PL)
KACZMAREK LESZEK (PL)
| AMENDED CLAIMS received by the International Bureau on 8 April 2015 (08.04.2015) 1. A genetically encoded MMP-9 activity biosensor that is anchored in the plasma membrane comprising the teal fluorescent protein mTFPl as a FRET donor fluorescent protein and two Venus Fluorescent Proteins as FRET acceptor fluorescent proteins all separated by flexible linkers, wherein the two Venus Fluorescent Proteins are separated by a flexible linker comprising seven repeats of GGSGSR hexapeptide, and one of the Venus Fluorescent Proteins is separated from the teal fluorescent protein mTFPl by an a-helical linker comprising a synthetic MMP-9 cleavage site or a linker comprising a synthetic MMP-9 cleavage site and only one GGTGGT hexapeptide. 2. The biosensor of claim 1, wherein one of the Venus Fluorescent Proteins is separated from the teal fluorescent protein mTFPl by α-helical linker comprising sequence EEEIRE AFRVFPRS LS LRH VMTNL. 3. The biosensor of one of claims 1 or 2, wherein the synthetic MMP-9 cleavage site corresponds to PRSLS sequence. 4. The biosensor of one of claims 1-3, wherein it is anchored in the plasma membrane by a PDGFR transmembrane domain. 5. Use of the genetically encoded MMP-9 activity biosensor defined in any of the claims 1- 4 as a system for investigation of the proteolytic activity of MMP-9 in vitro and in vivo in living cells. |