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Chapter category: Cell Metabolism

Cell-Free Synthesis of Defined Protein Conjugates by Site-Directed Cotranslational Labeling

This chapter appears in the following book:

Cell-Free Protein Expression

Edited by: Wieslaw Kudlicki, Federico Katzen and Robert Bennett
ISBN: 978-1-58706-123-3
» Get more information about this book at landesbioscience.com «

Chapter authors:
Michael Gerrits, Jan Strey, Iris Claußnitzer, Uritza von Groll, Frank Schäfer, Martina Rimmele and Wolfgang Stiege


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Proteins provided with unique functional groups such as affinity labels or fluorescence moieties offer high potential in many biotechnological or biomedical investigations, e.g., immobilization studies or high throughput screenings. An attractive alternative to known posttranslational methods of protein modification is the site-directed cotranslational incorporation of unnatural amino acids. Here we point out different aspects of this method with regard to the synthesis of protein conjugates bearing different functionalities. Moreover we describe a cell-free system enabling tRNA-based synthesis of modified proteins even with large functional groups in high yields. This specialized cell-free system contains a substantially decreased level of release factor 1 (RF1) and originates from an E. coli strain encoding a tagged RF1 variant. We present the efficient amber suppressor tRNA?mediated incorporation of the unnatural amino acid biocytin (biotinylated lysine) into stoichiometrically defined protein conjugates, containing the biotin label at the desired position. As potential applications we demonstrate the usefulness of the system for the immobilization of biotinylated proteins directly from a cell-free system and for interaction studies. Finally we show the cotranslational incorporation of a large fluorescent amino acid using our system.

Michael Gerrits
RiNA Netzwerk RNA Technologien GmbH

Jan Strey
RiNA Netzwerk RNA Technologien GmbH

Iris Claußnitzer
RiNA Netzwerk RNA Technologien GmbH

Uritza von Groll
QIAGEN GmbH

Frank Schäfer
QIAGEN GmbH

Martina Rimmele
RiNA Netzwerk RNA Technologien GmbH

Wolfgang Stiege
RiNA Netzwerk RNA Technologien GmbH

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