Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-15748
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Main Title: A CHO-Based Cell-Free Dual Fluorescence Reporter System for the Straightforward Assessment of Amber Suppression and scFv Functionality
Author(s): Krebs, Simon K.
Rakotoarinoro, Nathanaël
Stech, Marlitt
Zemella, Anne
Kubick, Stefan
Type: Article
URI: https://depositonce.tu-berlin.de/handle/11303/16969
http://dx.doi.org/10.14279/depositonce-15748
License: https://creativecommons.org/licenses/by/4.0/
Abstract: Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein’s coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyrCUA were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).
Subject(s): expanded genetic code
orthogonal system
noncanonical amino acid
unnatural amino acid
antibody
cell-free protein synthesis
mRFP1
sfGFP
Issue Date: 29-Apr-2022
Date Available: 20-May-2022
Language Code: en
DDC Class: 570 Biowissenschaften; Biologie
Sponsor/Funder: BMBF, 031B0078A, Basistechnologien: Zellfreie Systeme für die Funktionsanalyse transmembranärer Proteine (SysMem2016), Teilprojekt A
BMBF, 031B0831C, Maßgeschneiderte Inhaltsstoffe 2 - Verbundvorhaben: "CEFOX - Teilprojekt C"
Journal Title: Frontiers in Bioengineering and Biotechnology
Publisher: Frontiers
Volume: 10
Article Number: 873906
Publisher DOI: 10.3389/fbioe.2022.873906
EISSN: 2296-4185
TU Affiliation(s): Fak. 3 Prozesswissenschaften » Inst. Biotechnologie
Appears in Collections:Technische Universität Berlin » Publications

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