Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs.
We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D015723:Gene Library; D005816:Genetic Complementation Test / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014162:Transfection; D020798:Two-Hybrid System Techniques; D051981:Uracil-DNA Glycosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D053200:Fas-Associated Death Domain Protein; D011993:Recombinant Fusion Proteins; D051981:Uracil-DNA Glycosidase; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1