Simultaneous dual protein labeling using a triorthogonal reagent

Mohammad Rashidian, Sidath C Kumarapperuma, Kari Gabrielse, Adrian Fegan, Carston R Wagner, Mark D Distefano

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Construction of heterofunctional proteins is a rapidly emerging area of biotherapeutics. Combining a protein with other moieties, such as a targeting element, a toxic protein or small molecule, and a fluorophore or polyethylene glycol (PEG) group, can improve the specificity, functionality, potency, and pharmacokinetic profile of a protein. Protein farnesyl transferase (PFTase) is able to site-specifically and quantitatively prenylate proteins containing a C-terminal CaaX-box amino acid sequence with various modified isoprenoids. Here, we describe the design, synthesis, and application of a triorthogonal reagent, 1, that can be used to site-specifically incorporate an alkyne and aldehyde group simultaneously into a protein. To illustrate the capabilities of this approach, a protein was enzymatically modified with compound 1 followed by oxime ligation and click reaction to simultaneously incorporate an azido-tetramethylrhodamine (TAMRA) fluorophore and an aminooxy-PEG moiety. This was performed with both a model protein [green fluorescent protein (GFP)] as well as a therapeutically useful protein [ciliary neurotrophic factor (CNTF)]. Next, a protein was enzymatically modified with compound 1 followed by coupling to an azido-bis-methotrexate dimerizer and aminooxy-TAMRA. Incubation of that construct with a dihydrofolate reductase (DHFR)-DHFR-anti-CD3 fusion protein resulted in the self-assembly of nanoring structures that were endocytosed into T-leukemia cells and visualized therein. These results highlight how complex multifunctional protein assemblies can be prepared using this facile triorthogonal approach.

Original languageEnglish (US)
Pages (from-to)16388-16396
Number of pages9
JournalJournal of the American Chemical Society
Volume135
Issue number44
DOIs
StatePublished - Nov 6 2013

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Labeling
Proteins
Tetrahydrofolate Dehydrogenase
Fluorophores
Polyethylene glycols
Nanorings
Ciliary Neurotrophic Factor
T-Cell Leukemia
Pharmacokinetics
Oximes
Alkynes
Poisons
Terpenes
Transferases
Green Fluorescent Proteins
Aldehydes
Methotrexate
Endocytosis
Self assembly
Ligation

Cite this

Simultaneous dual protein labeling using a triorthogonal reagent. / Rashidian, Mohammad; Kumarapperuma, Sidath C; Gabrielse, Kari; Fegan, Adrian; Wagner, Carston R; Distefano, Mark D.

In: Journal of the American Chemical Society, Vol. 135, No. 44, 06.11.2013, p. 16388-16396.

Research output: Contribution to journalArticle

Rashidian, Mohammad ; Kumarapperuma, Sidath C ; Gabrielse, Kari ; Fegan, Adrian ; Wagner, Carston R ; Distefano, Mark D. / Simultaneous dual protein labeling using a triorthogonal reagent. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 44. pp. 16388-16396.
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