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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."

SUBMITTER: Otten C 

PROVIDER: S-EPMC4388811 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

Otten Christian C   De Benedetti Stefania S   Gaballah Ahmed A   Bühl Henrike H   Klöckner Anna A   Brauner Jarryd J   Sahl Hans-Georg HG   Henrichfreise Beate B  

PloS one 20150407 4


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.  ...[more]

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