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Bioconjug Chem. 2016 Jan 20;27(1):47-53. doi: 10.1021/acs.bioconjchem.5b00618. Epub 2015 Dec 21.

Selective Coupling of Click Anchors to Proteins via Trypsiligase.

Author information

1
Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.

Abstract

The combination of pure chemical methods with enzymatic approaches offers a kit system with maximum flexibility for site-specifically tagging proteins with a broad variety of artificial structures. Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has been used to introduce click anchors into the human protein cyclophilin 18 and the antibody Fab fragments anti-TNFα and anti-Her2. The subsequent click reactions with tetrazine or norbornene moieties lead to quantitative conversions to the corresponding dihydropyridazine products, thereby forming a stable covalent linkage between the label and the protein of interest. With this technology, cyclophilin 18 has been efficiently modified with the fluorescent dansyl moiety and the pharmaceutically relevant polymer PEG exclusively at its N-terminus. With the same methodology, the Fab fragments of anti-TNFα and anti-Her2 were derivatized exclusively at their C-terminal ends with PEG and the fluorescent dye carboxyfluorescein in the case of anti-TNFα or with the cytotoxic payload DM1 in the case of anti-Her2, to form a homogeneous antibody-drug conjugate (ADC).

PMID:
26670641
DOI:
10.1021/acs.bioconjchem.5b00618
[Indexed for MEDLINE]

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