Site-specific labeling of cellular proteins with unnatural substrates of biotin ligases

E. coli biotin ligase (BirA) catalyzes the site-specific ligation of biotin to the lysine within its 15-amino acid peptide substrate (AP). We harnessed the high peptide substrate specificity of BirA to develop a general methodology for site-specific protein labeling with biophysical probes. We synthesized an isosteric ketone analog of biotin and discovered that BirA also ligates it to the AP with similar kinetics while retaining the high peptide specificity of the native reaction. Because ketones are absent from native cell surfaces, recombinant AP-fused cell surface proteins can be tagged with the ketone analog, and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrated this two-step protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe were incorporated, with total labeling times as short as 20 minutes. To develop new protein labeling methodology, we synthesized azide analogs of biotin, but found that BirA did not accept them as substrates. Our efforts to rationally design mutations in the active site to accommodate these analogs met with little success. We thus investigated in vitro compartmentalization as a platform for re-engineering the substrate specificity of BirA through in vitro evolution.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Chen, Irwin

Advisor dc:contributor.advisor

• Alice Y. Ting.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/41555

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/41555