Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae
- 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 notWith 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.…
Verfasserangaben: | Hakan Dortay, Sandra M. Schmöckel, Jörg FettkeORCiDGND, Bernd Müller-RöberORCiDGND |
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DOI: | https://doi.org/10.1016/j.pep.2011.03.010 |
ISSN: | 1046-5928 |
Titel des übergeordneten Werks (Englisch): | Protein expression and purification |
Verlag: | Elsevier |
Verlagsort: | San Diego |
Publikationstyp: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Jahr der Erstveröffentlichung: | 2011 |
Erscheinungsjahr: | 2011 |
Datum der Freischaltung: | 26.03.2017 |
Freies Schlagwort / Tag: | C-reactive protein; In vitro expression; Leishmania; Protein expression; Protein purification |
Band: | 78 |
Ausgabe: | 1 |
Seitenanzahl: | 6 |
Erste Seite: | 55 |
Letzte Seite: | 60 |
Fördernde Institution: | German Ministry of Education and Research (BMBF) [FKZ 03IP515] |
Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
Peer Review: | Referiert |