Specific and Covalent Cross-Linking of Proteins In Vivo Using the SortaseA Enzyme

Genetic fusion proteins are a major contributor to recombinant biotechnology today.  These fusions can increase the solubility of poorly folded proteins, act as scaffolds for ligand binding regions or serve as affinity purification domains.  Although genetic fusions are typically soluble and active, many fusion products do not fold properly in cells.  During the protein folding process nascent proteins can interact with each other, often altering their natural folding process creating mis-folded, inactive and aggregated products.   Eukaryotic cells alleviate this problem by allowing the protein domains fold separately and to interact post-translationally.  We present a similar strategy for assembling fully folded and active recombinant proteins in E. coli through the covalent and post-translational cross-linking of the proteins via the SortaseA enzyme.  This method is thought to (1) alleviate the problems associated with folding interaction of genetic fusions, (2) link the proteins together irreversibly, (3) occur in vivo, (4) allow cross-linking without a significant energy burden to cells, and (5) apply universally to many proteins.

      Short peptide tags that are recognized by SortaseA were genetically added to the N- and C-termini of the desired protein targets.  The covalent fusion of the peptide tags and the attached proteins of interest occurred in vivo upon co-expression with the SortaseA enzyme.  We have used this technique of post-translational protein fusion to successfully cross-link a variety of proteins inside E. coli cells including fluorescent proteins (CFP and YFP), enzymes (beta-lactamase and enolase), and maltose binding protein.  This technology has implications for the creation of fusion proteins such as protease-resistant bifunctional antibodies that do not fold as genetic fusions, the specific linkage of enzymes to artificial supports as novel catalysts, the construction of fused multi-enzyme pathways, and the directed polymerization of proteins.