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. 2021:2305:175-190.
doi: 10.1007/978-1-0716-1406-8_9.

Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems

Marlitt Stech  1 , Nathanaël Rakotoarinoro  1   2 , Tamara Teichmann  1 , Anne Zemella  1 , Lena Thoring  1 , Stefan Kubick  3   4   5
Affiliations

Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems

Marlitt Stech et al. Methods Mol Biol. 2021.

Abstract

Cell-free protein synthesis (CFPS) enables the development of antibody conjugates, such as fluorophore conjugates and antibody-drug conjugates (ADCs), in a rapid and straightforward manner. In the first part, we describe the cell-free synthesis of antibodies containing fluorescent non-canonical amino acids (ncaa) by using pre-charged tRNA. In the second part, we describe the cell-free synthesis of antibodies containing ncaa by using an orthogonal system, followed by the site-specific conjugation of the fluorescent dye DyLight 650-phosphine. The expression of the antibodies containing ncaa was analyzed by SDS-PAGE, followed by autoradiography and the labeling by in-gel fluorescence. Two different fluorescently labeled antibodies could be generated.

Keywords: Antibody; Antibody conjugates; Cell-free protein synthesis; Conjugation; IgG1; Non-canonical amino acid.

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