@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{StechMerkSchenketal.2012,
  author    = {Stech, Marlitt and Merk, Helmut and Schenk, J{\"o}rg A. and St{\"o}cklein, Walter F. M. and W{\"u}stenhagen, Doreen Anja and Micheel, Burkhard and Duschl, Claus and Bier, Frank Fabian and Kubick, Stefan},
  title     = {Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system},
  series = {Journal of biotechnology},
  volume    = {164},
  journal   = {Journal of biotechnology},
  number    = {2},
  publisher = {Elsevier},
  address   = {Amsterdam},
  issn      = {0168-1656},
  doi       = {10.1016/j.jbiotec.2012.08.020},
  pages     = {220 -- 231},
  year      = {2012},
  abstract  = {Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.},
  language  = {en}
}
@phdthesis{Stech2014,
  author    = {Stech, Marlitt},
  title     = {Investigations on the cell-free synthesis of single-chain antibody fragments using a cukaryotic translation system},
  pages     = {ix, 126},
  year      = {2014},
  language  = {en}
}