The Trp cage motif as a scaffold for the display of a randomized peptide library on bacteriophage T7.
Phage libraries displaying linear or disulfide-constrained peptides often yield weak binders, upon screening against a target, and must be optimized to improve affinity. The disadvantages of libraries based on larger complex proteins, such as single chain antibodies, have stimulated interest in the development of smaller nonimmunoglobulin protein scaffolds. A promising candidate is the Trp cage motif, a 20-residue C-terminal sequence of exendin-4. Amino acid substitution within the Trp cage resulted in a 20-mer peptide recognized as an ultrafast cooperative folding miniprotein, with ideal characteristics for the discovery of small structured nonimmunoglobulin motifs having a stable tertiary structure. Although we were unable to display the Trp cage on M13 phage, successful display was achieved using the lytic T7 phage. Interestingly, mutations were observed at a frequency dependent on display valency. A Trp cage library designed with randomized amino acids at seven solvent-exposed positions was developed from 1.6 x 10(9) primary clones in T7Select10-3b. DNA sequencing of 109 library clones revealed 38% mutants and 16% truncations by TAG codons at randomized positions. Amino acid frequencies were largely within expected bounds and DIVAA analysis revealed that the library had an average diversity of 0.67. Utility of the library was demonstrated by identification of HPQ containing Trp cage miniproteins, which bound streptavidin, and AAADPYAQWLQSMGPHSGRPPPR, which bound to human bronchial epithelial cells. A high complexity library based on the Trp cage miniprotein has demonstrated potential for identifying novel cell and protein binding peptides that could be used for the delivery of therapeutic molecules or as target-specific therapeutic agents.
Herman
Richard E
RE
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021.
Badders
Douglas
D
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021.
Fuller
Mark
M
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021.
Makienko
Ekaterina G
EG
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021.
Houston
Michael E
ME
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021.
Quay
Steven C
SC
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021.
Johnson
Paul H
PH
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021. Electronic address: pjohnson@nastech.com.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001980:Bronchi; D002460:Cell Line; D004926:Escherichia coli / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D019151:Peptide Library; D010455:Peptides; C508725:Trp-cage peptide; D014764:Viral Proteins