Cell-free synthesis of cytochrome bo3 ubiquinol oxidase in artificial membranes

Ahu Arslan Yildiz, Wolfgang Knoll, Robert B. Gennis, Eva Kathrin Sinner

Research output: Contribution to journalArticle

Abstract

The analysis of membrane proteins is notoriously difficult because isolation and detergent-mediated reconstitution often results in compromising the protein structure and function. We introduce a novel strategy of combining a cell-free expression method for synthesis of a protein species coping with one of the most important obstacles in membrane protein research - preserving the structural-functional integrity of a membrane protein species and providing a stable matrix for application of analytical tools to characterize the membrane protein of interest. We address integration and subsequent characterization of the cytochrome bo3 ubiquinol oxidase (Cyt-bo3) from de novo synthesis without the effort of conventional cell culture, isolation, and purification procedures. The experimental output supports our idea of a suitable platform for in vitro protein synthesis and functional integration into a membrane-mimicking structure. We show the compatibility of different concepts of in vitro synthesis toward biosensor applicability by the example of Cyt-bo 3 protein expression. Our results obtained from in vitro synthesized proteins displayed similar behavior to proteins isolated from the cellular context. Overall, our approach is suitable for the in vitro expression of "complex" protein species such as Cyt-bo3, which can be reproducible and stably synthesized and preserved in robust, synthetic planar membrane architecture.

Original languageEnglish (US)
Pages (from-to)39-45
Number of pages7
JournalAnalytical Biochemistry
Volume423
Issue number1
DOIs
StatePublished - Apr 1 2012

Keywords

  • Artificial membrane
  • Cell-free protein synthesis
  • Electron transport chain
  • Mitochondrial disorders
  • Synthetic bioarchitectures

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Cell-free synthesis of cytochrome bo<sub>3</sub> ubiquinol oxidase in artificial membranes'. Together they form a unique fingerprint.

  • Cite this