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

  • Marlitt Stech
  • , Nathanaël Rakotoarinoro
  • , Tamara Teichmann
  • , Anne Zemella
  • , Lena Thoring
  • , Stefan Kubick*
  • *Corresponding author for this work

Research output: Book chapterChapterpeer-review

7 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationStructural Proteomics
Subtitle of host publicationHigh-Throughput Methods
EditorsRaymond J. Owens
PublisherHumana Press Inc.
Pages175-190
Number of pages16
ISBN (Electronic)978-1-0716-1406-8
ISBN (Print)978-1-0716-1405-1
DOIs
Publication statusPublished - 2021
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume2305
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Antibody
  • Antibody conjugates
  • Cell-free protein synthesis
  • Conjugation
  • IgG1
  • Non-canonical amino acid

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