@article{DortaySchmoeckelFettkeetal.2011,
  author    = {Dortay, Hakan and Schm{\"o}ckel, Sandra M. and Fettke, J{\"o}rg and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae},
  series = {Protein expression and purification},
  volume    = {78},
  journal   = {Protein expression and purification},
  number    = {1},
  publisher = {Elsevier},
  address   = {San Diego},
  issn      = {1046-5928},
  doi       = {10.1016/j.pep.2011.03.010},
  pages     = {55 -- 60},
  year      = {2011},
  abstract  = {With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca(2+)-dependent manner. In this report we verify the binding specificity of recombinant CRP from L tarentolae (2 mu g/mL culture medium) for PCh.},
  language  = {en}
}
@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}
}