Dötsch Lab

Goethe Universität Frankfurt

Research overview

Cell-Free Expression & Membrane Proteins

Project leader:

Frank Bernhard

  1. Cell-free expression platform at the IBPC
  2. Cell-free membrane protein production
  3. Co-translational insertion of membrane proteins into nanoparticles
  4. Characterization of cell-free sythesized GPCR/G-protein complexes
  5. Nanomembranes as transfer vectors for integrated membrane proteins
  6. Cell-free labelling of proteins

1. Cell-free expression platform at the IBPC

We use cell-free expression technology for the production of challenging proteins such as membrane proteins or toxins, as well as for the labelling of proteins with stable isotopes or fluorescent markers.
Our established in-house cell-free expression system comprises the production of cell-free lysates from special E. coli strains, the manufacturing of reaction containers, and the preparation of additives such as nanoparticles or enzymes.

Two-compartment cell-free expression configuration for preparative scale protein production
Cell-free reaction container for analytical and preparative scale expression

Our lysates are standardized and characterized by proteome analysis (Foshag 2018).

Most efficient with > 3 mg/mL protein production is the HY-S30 lysate (available at CUBE-Biotech).

The HS-S30 lysate is enriched in chaperones suitable for optimized protein folding.

Selected Publications

2. Cell-free membrane-protein production

Membrane proteins can be cell-free synthesized in three different basic modes. Which mode is most suitable for a given membrane protein is subject of screening.

With the D-CF and L-CF mode, the membrane protein is cotranslationally folded in presence of hydrophobic additives. In the P-CF mode, the precipitated membrane proteins may retain folded structures and could be detergent-solubilized without refolding procedures.

Structures of cell-free synthesized membrane proteins: Different views of D-CF synthesized proteorhodopsin (Reckel 2011), P-CF synthesized Diacyl-glycero-kinaseA (Boland et al., 2014), fully assembled D-CF synthesized ATP synthase (Matthies et al., 2011).

Selected Publications:

3. Co-translational insertion of membrane proteins into nanoparticles

A special L-CF strategy only possible by cell-free expression is the direct cotranslational insertion of nascent membrane proteins into provided nanomembranes. The insertion is translocon independent and avoids any contact with detergents. The resulting membrane protein/nanoparticle complexes can instantly be used for structural or functional studies.

Nanodiscs can be loaded with almost any lipid combination and allow the systematic screening of lipid effects on protein function (Henrich et al., 2015)

Three nanoparticle formation strategies are possible with nanodiscs or Salipro particles:

  • 1: Preforming;
  • 2: Coforming;
  • 3: Coexpression

(Levin et al, in revision)

Selected Publications:

4. Characterization of cell-free synthesized GPCR/G-protein complexes

G-protein coupled receptors are synthesized by cotranslational insertion into tailored nanodisc membranes without any contact to detergents. The GPCRs are full-length and functional in ligand binding and G-protein coupling.

Ligand binding profiling of the human endothelin A and B receptors. The GPCRs show characteristic ligand selectivity (Proverbio et al., 2013).

Negative stain and cryo-electron microscopy of cell-free synthesized GPCR/G-protein complexes in nanodiscs

(Köck et al., 2022)

Selected Publications:

5. Nanomembranes as transfer vectors for integrated membrane proteins

Membranes of nanodiscs or Salipro particles efficiently fuse with other membranes and inserted membrane proteins can thus even be transferred into membranes of living cells (Patriarchi et al, Sci Rep). This new technique allows simultaneous in vitro and in vivo assays with same sample batch.

Transfer of the ion pump KR2 from nanodiscs into solid supported membranes (Henrich 2017) and bacteriorhodopsin from nanodiscs into lipidic cubic phase for subsequent crystallization (Nikolaev et al., 2017).

Transfer of cell-free synthesized GPCR/nanodisc complexes into mammalian cell membranes. The transfer is fast, tuneable, non-toxic to the cells and independent from type of membrane protein and composition of the nanomembrane. Created with  BioRender.com.

Localization of transferred GPCR-mNG compared with endogenuous synthesized GPCR-mNG after transfection. Transferred GPCRs are active in ligand binding, G-protein coupling and interact with endogenuous binding partners.

(Umbach et al., 2022)

Selected Publications:

6. Cell-free labelling of proteins

Cell-free expression gives complete control over the amino acid pool in the reaction. It is therefore the most efficient system for fast labelling of proteins, either by full-labelling or by combinatorial-labelling strategies.

Combinatorial labelling of the putative multidrug transporter TehA (Trbovic et al., 2005).

Reduction of NMR spectral complexity by methyl labelling of amino acid residues, exemplified with the proton pump proteorhodopsin (Lazarova et al., 2018).

Improving of NMR spectral resolution by gradual transition of nanodiscs into bicelles. The integrated membrane proteins stay integrated into lipid environments and do not contact the detergent (Laguerre et al., 2016).

Selected Publications: