Browsing by Author "Elfving, Karoliina"

Catcher-protein and Tag-peptide originate from split CnaB domains of Gram-positive bacteria surface proteins, which are stabilized by spontaneous intramolecular isopeptide bonds formed between lysine and asparagine residues. However, there is a limited number of non-cross-reacting Catcher and Tag pairs available where the reaction occurs close to the diffusion limit, and which can be used in multiple fragment ligation to construct recombinant fusion proteins. Therefore, a new Catcher/Tag system – LplCatcher/LplTag – was developed in our group from CnaB domain of Lactobacillus plantarum. However, the ligation efficiency of this pair needs to be improved to expand the application possibilities. Therefore, there is a need for efficient library screening method, which allows to detect improved protein-peptide pairs where the covalent interaction takes place rapidly. In this study a new high-throughput in-vivo screening system was developed for visualizing the ligation of Catcher/Tag fusion proteins using splitFAST fluorogenic reporter system for detecting the phenotype, and Fluorescence-activated cell sorting (FACS) for separating the variants at single cell level based on fluorescence intensity. splitFAST is a system engineered by splitting a fluorescent protein named Fluorescence-Activating and absorption-Shifting Tag (FAST) into CFAST and NFAST. The system can be utilized in visualizing the protein interactions because once NFAST and CFAST associate, in the presence of a fluorogen, they form the active and highly fluorescent FAST protein. Herein, Catcher-protein was fused with CFAST and Tag-peptide with NFAST, which allowed detecting the Catcher-Tag ligation ratio based on fluorescence with splitFAST system. Next, a screening system was developed for detecting Catcher variants with improved ligation efficiency. The developed high-throughput screening system showed high potential since visualizing the protein ligation was possible, and hence the system could help in expanding the Catcher/Tag toolbox by allowing large mutant library analyzes.