Identification of epitope-like consensus motifs using mRNA display.
The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we determined the peptide sequences recognized by the trypsin active site, and by the anti-c-Myc antibody, 9E10. Here we describe the use of two peptide libraries of different diversities, one a constrained library based on the trypsin inhibitor EETI-II, where only the six residues in the first loop were randomized (6.4 x 10(7) possible sequences, 6.0 x 10(11) sequences in the library), the other a linear-peptide library with 27 randomized amino acids (1.3 x 10(35) possible sequences, 2 x 10(13) sequences in the library). The constrained library was screened against the natural target of wild-type EETI, bovine trypsin, and the linear library was screened against the anti-c-myc antibody, 9E10. The analysis of selected sequences revealed minimal consensus sequences of PR(I,L,V)L for the first loop of EETI-II and LISE for the 9E10 epitope. The wild-type sequences, PRILMR for the first loop of EETI-II and QKLISE for the 9E10 epitope, were selected with the highest frequency, and in each case the complete wild-type epitope was selected from the library.
Phylos Inc., 128 Spring St, Lexington, MA 02421, USA. rbaggio@phylos.com
Rick
R
Baggio
Petra
P
Burgstaller
Stephen P
SP
Hale
Andrew R
AR
Putney
Meghan
M
Lane
Dasa
D
Lipovsek
Martin C
MC
Wright
Richard W
RW
Roberts
Rihe
R
Liu
Jack W
JW
Szostak
Richard W
RW
Wagner
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002417:Cattle; D016384:Consensus Sequence; D017124:Conserved Sequence; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010940:Plant Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology; D011691:Puromycin / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014361:Trypsin Inhibitors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009841:Oligonucleotides; D010446:Peptide Fragments; D019151:Peptide Library; D010940:Plant Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D014361:Trypsin Inhibitors; C063950:trypsin inhibitor EETI II protein, plant; D011691:Puromycin; D014357:Trypsin