Activity-enhanced variant of hyeractive Sleeping Beauty transposase SB100X

Technology Description

SB transposase is composed of an N-terminal DNA binding domain (aa1-110) and a C-terminal catalytic domain (aa114-340), which are connected via a flexible linker that also harbors the nuclear localization signal (aa97-123). Since the discovery of the SB system, several mutations have been identified which improve the enzyme’s properties. These efforts culminated in the (until now) most active SB variant (--> SB100X), while other mutations led to e.g., alterations of the enzyme’s stability, solubility, or integration profile (--> K248R). Most mariner transposases carry a WVPHEL motif (aa119-124), which has been shown to play an important role in regulating the transposase activity. SB and some other transposases, in contrast, contain an KKPLL motif instead, indicating that activity modulating mutations that work for WVPHEL-type transposases do not work for SB transposases. Surprisingly, the inventors found that substituting the glutamine residue right next to the KKPLL motif with cysteine (Q124C) facilitates (compared to SB100X) a robust about two-fold increase in transpositional activity. Importantly, the corresponding SB100X variant also shows resistance to the known overproduction inhibition, and the mutation is fully compatible with other clinically relevant SB transposase variants, including the recently identified safety improved SB100X mutant K248R (see TO 03-00541 and Figure).