@article{Pandey2023,
  author    = {Pandey, Yogesh},
  title     = {Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5.},
  series = {Biochimica et Biophysica Acta (BBA) - Biomembranes},
  volume    = {1865},
  journal   = {Biochimica et Biophysica Acta (BBA) - Biomembranes},
  number    = {5},
  publisher = {Elsevier},
  address   = {Amsterdam},
  issn      = {1879-2642},
  doi       = {10.1016/j.bbamem.2023.184144},
  year      = {2023},
  abstract  = {Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.},
  language  = {en}
}
@article{BroedelRaymondDumanetal.2013,
  author    = {Broedel, A. K. and Raymond, J. A. and Duman, J. G. and Bier, Frank Fabian and Kubick, S.},
  title     = {Functional evaluation of candidate ice structuring proteins using cell-free expression systems},
  series = {JOURNAL OF BIOTECHNOLOGY},
  volume    = {163},
  journal   = {JOURNAL OF BIOTECHNOLOGY},
  number    = {3},
  publisher = {ELSEVIER SCIENCE BV},
  address   = {AMSTERDAM},
  issn      = {0168-1656},
  doi       = {10.1016/j.jbiotec.2012.11.001},
  pages     = {301 -- 310},
  year      = {2013},
  abstract  = {Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications. (C) 2012 Elsevier B.V. All rights reserved.},
  language  = {en}
}