Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.
Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.
Department of Cell-free and Cell-based Bioproduction, Branch Bioanalysis and Bioprocesses, Fraunhofer-Institute for Cell Therapy and Immunology (IZI-BB), Potsdam-Golm, Germany.|Institute for Biotechnology, Technical University of Berlin (TUB), Gustav-Meyer-Allee 25, 13355, Berlin.
Lena
L
Thoring
Department of Cell-free and Cell-based Bioproduction, Branch Bioanalysis and Bioprocesses, Fraunhofer-Institute for Cell Therapy and Immunology (IZI-BB), Potsdam-Golm, Germany.
Doreen A
DA
Wüstenhagen
Department of Cell-free and Cell-based Bioproduction, Branch Bioanalysis and Bioprocesses, Fraunhofer-Institute for Cell Therapy and Immunology (IZI-BB), Potsdam-Golm, Germany.
Maria
M
Borowiak
Department of Cell-free and Cell-based Bioproduction, Branch Bioanalysis and Bioprocesses, Fraunhofer-Institute for Cell Therapy and Immunology (IZI-BB), Potsdam-Golm, Germany.
Marlitt
M
Stech
Department of Cell-free and Cell-based Bioproduction, Branch Bioanalysis and Bioprocesses, Fraunhofer-Institute for Cell Therapy and Immunology (IZI-BB), Potsdam-Golm, Germany.|Institute for Biotechnology, Technical University of Berlin (TUB), Gustav-Meyer-Allee 25, 13355, Berlin.
Andrei
A
Sonnabend
Department of Cell-free and Cell-based Bioproduction, Branch Bioanalysis and Bioprocesses, Fraunhofer-Institute for Cell Therapy and Immunology (IZI-BB), Potsdam-Golm, Germany.
Stefan
S
Kubick
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