dc.contributor.author	Krebs, Simon K.
dc.contributor.author	Rakotoarinoro, Nathanaël
dc.contributor.author	Stech, Marlitt
dc.contributor.author	Zemella, Anne
dc.contributor.author	Kubick, Stefan
dc.date.accessioned	2022-05-20T11:41:17Z
dc.date.available	2022-05-20T11:41:17Z
dc.date.issued	2022-04-29
dc.date.updated	2022-05-17T21:38:31Z
dc.description.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).	en
dc.description.sponsorship	BMBF, 031B0078A, Basistechnologien: Zellfreie Systeme für die Funktionsanalyse transmembranärer Proteine (SysMem2016), Teilprojekt A	en
dc.description.sponsorship	BMBF, 031B0831C, Maßgeschneiderte Inhaltsstoffe 2 - Verbundvorhaben: "CEFOX - Teilprojekt C"	en
dc.identifier.eissn	2296-4185
dc.identifier.uri	https://depositonce.tu-berlin.de/handle/11303/16969
dc.identifier.uri	http://dx.doi.org/10.14279/depositonce-15748
dc.language.iso	en	en
dc.rights.uri	https://creativecommons.org/licenses/by/4.0/	en
dc.subject.ddc	570 Biowissenschaften; Biologie	de
dc.subject.other	expanded genetic code	en
dc.subject.other	orthogonal system	en
dc.subject.other	noncanonical amino acid	en
dc.subject.other	unnatural amino acid	en
dc.subject.other	antibody	en
dc.subject.other	cell-free protein synthesis	en
dc.subject.other	mRFP1	en
dc.subject.other	sfGFP	en
dc.title	A CHO-Based Cell-Free Dual Fluorescence Reporter System for the Straightforward Assessment of Amber Suppression and scFv Functionality	en
dc.type	Article	en
dc.type.version	publishedVersion	en
dcterms.bibliographicCitation.articlenumber	873906	en
dcterms.bibliographicCitation.doi	10.3389/fbioe.2022.873906	en
dcterms.bibliographicCitation.journaltitle	Frontiers in Bioengineering and Biotechnology	en
dcterms.bibliographicCitation.originalpublishername	Frontiers	en
dcterms.bibliographicCitation.originalpublisherplace	Lausanne	en
dcterms.bibliographicCitation.volume	10	en
tub.accessrights.dnb	free	en
tub.affiliation	Fak. 3 Prozesswissenschaften::Inst. Biotechnologie	de
tub.affiliation.faculty	Fak. 3 Prozesswissenschaften	de
tub.affiliation.institute	Inst. Biotechnologie	de
tub.publisher.universityorinstitution	Technische Universität Berlin	en

A CHO-Based Cell-Free Dual Fluorescence Reporter System for the Straightforward Assessment of Amber Suppression and scFv Functionality

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