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Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism.

Abstract：

: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.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Waraho D,DeLisa MP

doi

10.1073/pnas.0704048106

subject

Has Abstract

pub_date

2009-03-10 00:00:00

pages

3692-7

issue

eissn

0027-8424

issn

1091-6490

pii

0704048106

journal_volume

106

pub_type

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Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism.