Second-generation covalent TMP-tag for live cell imaging.

Journal of the American Chemical Society (2012-08-10)
Zhixing Chen, Chaoran Jing, Sarah S Gallagher, Michael P Sheetz, Virginia W Cornish
ABSTRACT

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 655, BioReagent, suitable for fluorescence, ≥85% (HPLC)
Sigma-Aldrich
Atto 655 NHS ester, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 655-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 655 amine, suitable for fluorescence
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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