@article{SchlosshauerCavakZemellaetal.2022,
  author    = {Schloßhauer, Jeffrey and Cavak, Ni{\~n}o and Zemella, Anne and Thoring, Lena and Kubick, Stefan},
  title     = {Cell engineering and cultivation of chinese hamster ovary cells for the development of orthogonal eukaryotic cell-free translation systems},
  series = {Frontiers in molecular biosciences},
  volume    = {9},
  journal   = {Frontiers in molecular biosciences},
  publisher = {Frontiers Media},
  address   = {Lausanne},
  issn      = {2296-889X},
  doi       = {10.3389/fmolb.2022.832379},
  pages     = {13},
  year      = {2022},
  abstract  = {The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase.},
  language  = {en}
}
@article{KrebsRakotoarinoroStechetal.2022,
  author    = {Krebs, Simon K. and Rakotoarinoro, Nathanael and Stech, Marlitt and Zemella, Anne and Kubick, Stefan},
  title     = {A CHO-based cell-free dual fluorescence reporter system for the straightforward assessment of amber suppression and scFv functionality},
  series = {Frontiers in Bioengineering and Biotechnology},
  volume    = {10},
  journal   = {Frontiers in Bioengineering and Biotechnology},
  publisher = {Frontiers Media},
  address   = {Lausanne},
  issn      = {2296-4185},
  doi       = {10.3389/fbioe.2022.873906},
  pages     = {15},
  year      = {2022},
  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 tRNATyr(CUA) 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).},
  language  = {en}
}
@article{HaueisStechKubick2022,
  author    = {Haueis, Lisa and Stech, Marlitt and Kubick, Stefan},
  title     = {A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies},
  series = {Frontiers in Bioengineering and Biotechnology},
  volume    = {10},
  journal   = {Frontiers in Bioengineering and Biotechnology},
  publisher = {Frontiers Media},
  address   = {Lausanne},
  issn      = {2296-4185},
  doi       = {10.3389/fbioe.2022.896763},
  pages     = {11},
  year      = {2022},
  abstract  = {Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, similar to 15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation.},
  language  = {en}
}
@article{DondapatiLuebberdingZemellaetal.2019,
  author    = {Dondapati, Srujan Kumar and L{\"u}bberding, Henning and Zemella, Anne and Thoring, Lena and W{\"u}stenhagen, Doreen Anja and Kubick, Stefan},
  title     = {Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems},
  series = {Frontiers in pharmacology},
  volume    = {10},
  journal   = {Frontiers in pharmacology},
  publisher = {Frontiers Research Foundation},
  address   = {Lausanne},
  issn      = {1663-9812},
  doi       = {10.3389/fphar.2019.00917},
  pages     = {9},
  year      = {2019},
  abstract  = {Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.},
  language  = {en}
}