Protease specific for a novel sumo-derived protease cleavage site

Abstract

The present invention relates to a fusion protein, comprising the structure N-PCS-degSig-M-PCS-degSig-C; wherein N represents the N-terminus; PCSand PCSeach represent a protease cleavage site (PCS), which differ from each other in at least one amino acid residue: degSigrepresents a degradation signal which promotes degradation of the fusion protein in a host cell if PCSis cleaved by a protease such that the first amino acid of degSigbecomes the new N-terminus of the remaining fusion; M represents a cytoplasmic selection marker; and degSigrepresents a second degradation signal which promotes degradation of the fusion protein in a host cell if PCSis not cleaved by a protease; and C represents the C-terminus. Further provided is a nucleic acid construct, comprising a nucleic acid sequence coding for said fusion protein, a nucleic acid expression construct library, comprising a plurality of such nucleic acid expression constructs in diversified form, and methods using the fusion protein and nucleic acid constructs coding therefor. Finally, the present invention provides variants of bdSUMO and bdSENP1 which have been identified by the methods of the present disclosure, and which exhibit improved properties over existing orthogonal protease/protease cleavage site-pairs which are currently used with wild-type bdSUMO and wildtype bdSENP1.