title: Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae.
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 μg/mL culture medium) for PCh.
authors: University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Strasse 24-25, Haus 20, 14476 Potsdam-Golm, Germany. dortay@uni-potsdam.de
authors: Hakan
authors: H
authors: Dortay
authors: Sandra M
authors: SM
authors: Schmöckel
authors: Joerg
authors: J
authors: Fettke
authors: Bernd
authors: B
authors: Mueller-Roeber
mesh_terms: D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D015153:Blotting, Western; D002097:C-Reactive Protein / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007716:Kluyveromyces / Q000235:genetics; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002097:C-Reactive Protein