Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase - PubMed (original) (raw)
doi: 10.1038/nmeth735. Epub 2005 Jan 21.
Affiliations
- PMID: 15782206
- DOI: 10.1038/nmeth735
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
Irwin Chen et al. Nat Methods. 2005 Feb.
Abstract
We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.
Similar articles
- Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase.
Stachler MD, Chen I, Ting AY, Bartlett JS. Stachler MD, et al. Mol Ther. 2008 Aug;16(8):1467-73. doi: 10.1038/mt.2008.129. Epub 2008 Jun 17. Mol Ther. 2008. PMID: 18560418 - Targeting quantum dots to surface proteins in living cells with biotin ligase.
Howarth M, Takao K, Hayashi Y, Ting AY. Howarth M, et al. Proc Natl Acad Sci U S A. 2005 May 24;102(21):7583-8. doi: 10.1073/pnas.0503125102. Epub 2005 May 16. Proc Natl Acad Sci U S A. 2005. PMID: 15897449 Free PMC article. - Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.
Howarth M, Ting AY. Howarth M, et al. Nat Protoc. 2008;3(3):534-45. doi: 10.1038/nprot.2008.20. Nat Protoc. 2008. PMID: 18323822 Free PMC article. - Proteomic navigation using proximity-labeling.
Gentzel M, Pardo M, Subramaniam S, Stewart AF, Choudhary JS. Gentzel M, et al. Methods. 2019 Jul 15;164-165:67-72. doi: 10.1016/j.ymeth.2019.03.028. Epub 2019 Apr 4. Methods. 2019. PMID: 30953756 Review. - Molecular biology of biotin attachment to proteins.
Chapman-Smith A, Cronan JE Jr. Chapman-Smith A, et al. J Nutr. 1999 Feb;129(2S Suppl):477S-484S. doi: 10.1093/jn/129.2.477S. J Nutr. 1999. PMID: 10064313 Review.
Cited by
- Cell-surface sensors: lighting the cellular environment.
Ali MM, Kang DK, Tsang K, Fu M, Karp JM, Zhao W. Ali MM, et al. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2012 Sep-Oct;4(5):547-61. doi: 10.1002/wnan.1179. Epub 2012 Jul 3. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2012. PMID: 22761045 Free PMC article. Review. - Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.
Sletten EM, Bertozzi CR. Sletten EM, et al. Angew Chem Int Ed Engl. 2009;48(38):6974-98. doi: 10.1002/anie.200900942. Angew Chem Int Ed Engl. 2009. PMID: 19714693 Free PMC article. - A monovalent streptavidin with a single femtomolar biotin binding site.
Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A, Ting AY. Howarth M, et al. Nat Methods. 2006 Apr;3(4):267-73. doi: 10.1038/nmeth861. Nat Methods. 2006. PMID: 16554831 Free PMC article. - Novel peptide sequence ("IQ-tag") with high affinity for NIR fluorochromes allows protein and cell specific labeling for in vivo imaging.
Kelly KA, Carson J, McCarthy JR, Weissleder R. Kelly KA, et al. PLoS One. 2007 Jul 25;2(7):e665. doi: 10.1371/journal.pone.0000665. PLoS One. 2007. PMID: 17653285 Free PMC article. - Discovery of Potent and Selective Inhibitors against Protein-Derived Electrophilic Cofactors.
Wang X, Lin Z, Bustin KA, McKnight NR, Parsons WH, Matthews ML. Wang X, et al. J Am Chem Soc. 2022 Mar 30;144(12):5377-5388. doi: 10.1021/jacs.1c12748. Epub 2022 Mar 2. J Am Chem Soc. 2022. PMID: 35235319 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous