Using phage display to select antibodies recognizing post-translational modifications independently of sequence context.
Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.
Kehoe
John W
JW
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and Department of Anesthesia and Pharmaceutical Chemistry, San Francisco General Hospital 94110, USA.
Velappan
Nileena
N
Walbolt
Monica
M
Rasmussen
Jytte
J
King
Dave
D
Lou
Jianlong
J
Knopp
Kristeene
K
Pavlik
Peter
P
Marks
James D
JD
Bertozzi
Carolyn R
CR
Bradbury
Andrew R M
AR
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D002417:Cattle; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification / Q000378:metabolism; D007865:Leeches; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000276:immunology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library; C005662:tyrosine O-sulfate; D014443:Tyrosine