Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

John W Kehoe,Monica Walbolt,Dave King,Peter Pavlik,James D Marks,Carolyn R Bertozzi,Jytte Rasmussen,Jianlong Lou,Nileena Velappan,Kristeene Knopp,Andrew R.M Bradbury

doi:10.1074/mcp.m600314-mcp200

Abstract

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.

Highlights

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
It is possible that some post-translational modifications, such as phosphotyrosine, are intrinsically more antigenic either because the modified side chain is further from the peptide backbone than, for example, phosphoserine, or because substituted aromatic residues are in general more antigenic than modified residues that resemble metabolic intermediates
The tyrosine sulfated chemokine receptors CCR5, CXCR4, and CCR2 rely on their sulfate groups to increase binding affinity for both chemokines and human immunodeficiency virus (28 –32) as do the tyrosine sulfated complementarity-determining regions of some human immunodeficiency virus-neutralizing antibodies, the efficacy of which is reduced with elimination of this post-translational modifications (PTMs) [33, 34]

Summary

EXPERIMENTAL PROCEDURES

Synthesis of the Antigens—Pep and Pep were synthesized on an Applied Biosystems 431A peptide synthesizer at 0.1 mmol scale using standard dicyclohexylcarbodiimide (Novabiochem) couplings with N-hydroxybenzotriazole (Novabiochem). The cells were incubated at 4 °C for 15 min and pelleted, and 10 ␮l of the supernatant was added to the 80 ␮l of previously collected induction medium This crude prep contains scFv released into the growth supernatant as well as that retained in the periplasm. The scFv fragments were allowed to incubate at room temperature for 90 min before removal and washing with 200 ␮l/well PBS twice. 100 ␮l of SV5 diluted 1:2000 in PBS with 1% fish gelatin was added, and after 1 h at room temperature, the antibody solution was removed, and the wells were washed twice with 200 ␮l/well PBS. After 1 h at room temperature the plate was washed with PBS three times, and 100 ␮l of the partially purified scFv solution was added to each well for 1.5 h. The absorbance at 405 nm reported represents the final value obtained after background subtraction

RESULTS

No picked

DISCUSSION