Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments.
We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude. beta-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.
Wehrman
Tom
T
Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Kleaveland
Benjamin
B
Her
Jeng-Horng
JH
Balint
Robert F
RF
Blau
Helen M
HM
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005816:Genetic Complementation Test; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D010446:Peptide Fragments; D010455:Peptides; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases