Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Paul J. Focke, Christopher Hein, Beate Hoffmann, Kimberly Matulef, Frank Bernhard, Volker Dötsch, Francis I. Valiyaveetil

Research output: Research - peer-reviewArticle

  • 3 Citations

Abstract

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K+ channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

LanguageEnglish (US)
Pages4212-4219
Number of pages8
JournalBiochemistry
Volume55
Issue number30
DOIs
StatePublished - Aug 2 2016

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Membrane Proteins
Proteins
In Vitro Techniques
Precipitates
Amino Acid Transport Systems
Lipids
Peptides
Machinery
Crystal structure
Topology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Focke, P. J., Hein, C., Hoffmann, B., Matulef, K., Bernhard, F., Dötsch, V., & Valiyaveetil, F. I. (2016). Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression. Biochemistry, 55(30), 4212-4219. DOI: 10.1021/acs.biochem.6b00488

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression. / Focke, Paul J.; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I.

In: Biochemistry, Vol. 55, No. 30, 02.08.2016, p. 4212-4219.

Research output: Research - peer-reviewArticle

Focke, PJ, Hein, C, Hoffmann, B, Matulef, K, Bernhard, F, Dötsch, V & Valiyaveetil, FI 2016, 'Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression' Biochemistry, vol 55, no. 30, pp. 4212-4219. DOI: 10.1021/acs.biochem.6b00488
Focke PJ, Hein C, Hoffmann B, Matulef K, Bernhard F, Dötsch V et al. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression. Biochemistry. 2016 Aug 2;55(30):4212-4219. Available from, DOI: 10.1021/acs.biochem.6b00488
Focke, Paul J. ; Hein, Christopher ; Hoffmann, Beate ; Matulef, Kimberly ; Bernhard, Frank ; Dötsch, Volker ; Valiyaveetil, Francis I./ Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression. In: Biochemistry. 2016 ; Vol. 55, No. 30. pp. 4212-4219
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