Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism.
We have developed a reliable genetic selection strategy for isolating interacting proteins based on the "hitchhiker" mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate noncovalent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an N-terminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 beta-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer beta-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.
Waraho
Dujduan
D
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
DeLisa
Matthew P
MP
D000469:Alkaline Phosphatase / Q000378:metabolism; D000907:Antibodies, Bacterial / Q000378:metabolism; D000942:Antigens, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005057:Eukaryotic Cells; D007135:Immunoglobulin Variable Region; D042541:Intracellular Space / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D019897:Periplasm / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D021381:Protein Transport; D012641:Selection, Genetic
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli