Cell-free synthesis of cytochrome bo 3 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 bo 3 ubiquinol oxidase (Cyt-bo 3) 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-bo 3, 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

Fingerprint

Artificial Membranes
Cytochromes
Membranes
Membrane Proteins
Proteins
Membrane structures
Cell Separation
Biosensing Techniques
ubiquinol oxidase
cytochrome o oxidase
Cell culture
Biosensors
Detergents
Purification
Cell Culture Techniques
In Vitro Techniques
Research

Keywords

  • Artificial membrane
  • Cell-free protein synthesis
  • Cytochrome bo ubiquinol oxidase
  • Electron transport chain
  • Mitochondrial disorders
  • Synthetic bioarchitectures

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cell-free synthesis of cytochrome bo 3 ubiquinol oxidase in artificial membranes. / Yildiz, Ahu Arslan; Knoll, Wolfgang; Gennis, Robert B.; Sinner, Eva Kathrin.

In: Analytical Biochemistry, Vol. 423, No. 1, 01.04.2012, p. 39-45.

Research output: Contribution to journalArticle

Yildiz, Ahu Arslan ; Knoll, Wolfgang ; Gennis, Robert B. ; Sinner, Eva Kathrin. / Cell-free synthesis of cytochrome bo 3 ubiquinol oxidase in artificial membranes. In: Analytical Biochemistry. 2012 ; Vol. 423, No. 1. pp. 39-45.
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