@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{DortayAkulaWestphaletal.2011,
  author    = {Dortay, Hakan and Akula, Usha Madhuri and Westphal, Christin and Sittig, Marie and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {High-throughput protein expression using a combination of ligation-independent cloning (LIC) and infrared fluorescent protein (IFP) detection},
  series = {PLoS one},
  volume    = {6},
  journal   = {PLoS one},
  number    = {4},
  publisher = {PLoS},
  address   = {San Fransisco},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0018900},
  pages     = {19},
  year      = {2011},
  abstract  = {Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here.},
  language  = {en}
}