Targeted modification and transportation of cellular proteins.
Peptide aptamers are proteins selected from combinatorial libraries that display conformationally constrained variable regions. Peptide aptamers can disrupt specific protein interactions and thus represent a useful method for manipulating protein function in vivo. Here, we describe aptamer derivatives that extend the range of functional manipulations. We isolated an aptamer with increased affinity for its Cdk2 target by mutagenizing an existing aptamer and identifying tighter binding mutants with calibrated two-hybrid reporter genes. We used this and other anti-Cdk2 aptamers as recognition domains in chimeric proteins that contained other functional moieties. Aptamers fused to the catalytic domain of a ubiquitin ligase specifically decorated LexA-Cdk2 with ubiquitin moieties in vivo. Aptamers against Cdk2 and another protein, Ste5, that carried a nuclear localization sequence transported their targets into the nucleus. These experiments indicate that fusion proteins containing aptameric recognition moieties will be useful for specific modification of protein function in vivo.
Colas
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P
Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France.
Cohen
B
B
Ko Ferrigno
P
P
Silver
P A
PA
Brent
R
R
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D017931:DNA Primers; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D017931:DNA Primers; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein