A bacterial two-hybrid system based on the twin-arginine transporter pathway of E. coli.
We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.
Strauch
Eva-Maria
EM
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Georgiou
George
G
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; C408585:twin-arginine translocase complex, E coli; D019704:Protein Disulfide-Isomerases