title: Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.
abstract: Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Christian
authors: C
authors: Otten
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Stefania
authors: S
authors: De Benedetti
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Ahmed
authors: A
authors: Gaballah
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Henrike
authors: H
authors: Bühl
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Anna
authors: A
authors: Klöckner
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Jarryd
authors: J
authors: Brauner
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Hans-Georg
authors: HG
authors: Sahl
authors: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
authors: Beate
authors: B
authors: Henrichfreise
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001622:Betaine / Q000737:chemistry; D020134:Catalytic Domain; D002689:Chlamydia / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D012997:Solvents; D001622:Betaine