Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2.
A network of interacting proteins controls the activity of cyclin-dependent kinase 2 (Cdk2) (refs 1,2) and governs the entry of higher eukaryotic cells into S phase. Analysis of this and other genetic regulatory networks would be facilitated by intracellular reagents that recognize specific targets and inhibit specific network connections. We report here the expression of a combinatorial library of constrained 20-residue peptides displayed by the active-site loop of Escherichia coli thioredoxin, and the use of a two-hybrid system to select those that bind human Cdk2. These peptide aptamers were designed to mimic the recognition function of the complementarity-determining regions of immunoglobulins. The aptamers recognized different epitopes on the Cdk2 surface with equilibrium dissociation constant in the nanomolar range; those tested inhibited Cdk2 activity. Our results show that peptide aptamers bear some analogies with monoclonal antibodies, with the advantages that they are isolated together with their coding genes, that their small size should allow their structures to be solved, and that they are designated to function inside cells.
Colas
P
P
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Cohen
B
B
Jessen
T
T
Grishina
I
I
McCoy
J
J
Brent
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004926:Escherichia coli; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D013879:Thioredoxins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases