Epitope selection from an uncensored peptide library displayed on avian leukosis virus.
Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10(6) different members was generated from a plasmid library of approximately 5 x 10(6) diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries.
Molecular Medicine Program, Mayo Clinic Rochester, Rochester, MN 55905, USA.
Pranay D
PD
Khare
Ana G
AG
Rosales
Kent R
KR
Bailey
Stephen J
SJ
Russell
Mark J
MJ
Federspiel
D000595:Amino Acid Sequence; D000818:Animals; D001354:Avian Leukosis Virus / Q000235:genetics*; D002460:Cell Line; D002645:Chickens; D000939:Epitopes; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids
D000939:Epitopes; D019151:Peptide Library