6th P4EU Meeting Vienna

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1 6th P4EU Meeting Vienna Cell-free production P2 (Dötsch/Bernhard): of membrane proteins Cell-free expression of GPCRs: In vitro characterization of the endothelin receptor signal perception and transduction Frank Bernhard Institute of Biophysical Chemistry University of Frankfurt, Germany with: P15/Meier,..

2 Bottlenecks in the Production of Membrane Proteins Limited production pipelines Specific targeting & translocation pathways needed Inclusion bodies and proteolysis Membrane space limited Functional membrane insertion can cause toxic effects

3 Cellular / Cell-Free Membrane Protein Expression Transformation Cloning/DNA preparation / linear PCR fragments Reaction/Fermentation set-up Incubation (overnight days) Induction of expression Harvest Cell-disruption Isolation of membrane fraction Extraction of MPs out of membrane Targeting Translocation Toxicity Proteolysis Vector issues Cell handling Media Sterility Fusions Promoter Regulation/Induction Reaction set-up Incubation (overnight) + efficient labelling + individualized protocols + artificial hydrophobic environments (Solubilization 1-3 hrs) Cellular Purification Cell-free

4 Synthetic Biology / Cell-Free Systems - Bacterial S30 extract Removal of endogenous amino acids Elimination of endogenous mrna - T7-RNA polymerase Template DNA (T7-regulatory sequences), e.g. pet-vectors, pivex - Precursors (NTPs, amino acids, energy precursors, trnas ) Optional additives: - Efficiency enhancer: PEG, oxalate - Protein chaperones: GroEL/ES - Chemical chaperones: Polysaccharides, alcohols - Target specific stabilizers: Ligands, cofactors - Solubilization compounds: Detergents, lipids - Redox systems: DTT, glutathione system Reduced complexity Manipulation of cell background Direct control of expression machinery Designed environments Re-design of membrane protein production pathways

5 Positron Research Group B. Pucci / A. Polidori J. L. Popot D-CF (P-CF) L-CF

6 Analytical & Preparative Scale Reaction Containers Mini-CECF-Reactor Maxi-CECF-Reactor Reaction Mix (RM) Reaction Mix (RM) Feeding Mix (FM) Feeding Mix (FM) Schwarz et al (2007) Nat Protocols Schneider et al (2010) Methods Mol Biol Kai et al (2011) Methods Mol Biol Kai et al (2013) PlosOne Roos et al (2014) Methods Mol Biol + Shaker + Chemicals + Lysate

7 Systematic 3-Level Protocol Development I: Yield = Basic expression protocol: P-CF Basic parameters: Ions, extract source, energy system, template quality Tag variation Synthetic genes Type I aggregates Junge et al., (2011) New Biotechnology 28: Schwarz et al., (2008) Proteomics 8: Junge et al., (2008) Cell. Mol. Life Sci. 65:

8 Tag-Variation with GPCRs T7 Promotor N 80 RBS Expression tag 1 Coding sequence Name Nucleotide sequence Amino acid sequence AT (%) AT AAATAT TATAAATATTAT KYYKYY 100 SER AAATCATCATCATCATCA KSSSSS 72 H AAACCATACGATGGTCCA KPYDGP 55 G AAAAGTAAAGGAGAA GAA KSKGEE 72 Haberstock et al (2012) Protein Expr Purif

9 130 Targets 80 % success, Size: > 100 kda, 15 TMS Schwarz et al. (2010) Proteomics 10:

10 P-CF: Eukaryotic Organic Cation/Anion Transporters roct1 48 K m (MPP + ) = 35 µm (30 µm*) turnover number = 19 sec -1 (20 sec -1 *) MPP:1-methyl-4-phenyl-pyridinium Cation selectivity of roct1 IC50: TEA+: TPeA+: TBuA+: P-CF 159 µm / 196 µm* 2.9 µm / 1.8 µm* 20 µm / 19 µm* *roct1 from insect cells roat1 Keller et al. (2008) Biochemistry 47: Keller et al., (2011) J. Biol. Chem. 286:

11 P-CF: Channels and Porins Cell-free / native source PorA / PorH PorA-M / PorH-M PorA-M / PorH PorA / PorH-M inactive active active inactive Channels Von Bülow et al (2012) J Biol Chem Rath et al (2011) J Biol Chem Kai et al (2010) PlosOne

12 P-CF: NMR Structure of y-secretase Subunit Stefer et al., (2011) Science 333 Sobhanifar et al. (2010), PNAS 107 Sobhanifar et al. (2010), J Bio NMR 46 Reckel et al. (2008), PNAS 105 Koglin et al. (2006), Magn. Reson. 44 Trbovic et al., (2005), J.A.C.S. 127

13 P-CF: Crystallization 50 µm Boland et al (2014) CMLS

14 Crystal Structure of DgkA Li et al (2013) Nature E. coli enzyme, Mg 2+ -ATP-dependent phosphorylation of diacylglycerol 121 amino acids Homo-trimer, 3 transmembrane segments + 1 N-terminal amphiphilic helix LCP in meso, short chain 7.8 MAGs, 4 0 C, Is crystallization possible from cell-free expressed DgkA?

15 Spectroscopy DgkA in micelles A: UV-visible absorption spectra B: Electronic excitation spectra (normalized) Fluorescence yield of CF protein was 81% More polar environment of Trp and Tyr chromophores? C: Circular dichroism spectra 92% α-helix, 0.2% ß-strand, 8% random coil

16 Cell-free Versus Cellular DgkA 0,8 0,9 0,6 0,6 A 280 0,4 A 340 0, Elution volume (ml) 0,3 0 Coupled spectrophotometric assay E. coli DgkA: solid line cell-free DgkA: dashed line Time (min) A µg kda B kda µg Trimer: 28 kda Monomer: 13 kda Cell-free E. coli 16

17 Proof of Principle: LCP Crystallization of DgkA Cell-free production protocol: - 24 ml P-CF expression; 16 h - Centrifugation, washing; 1 h - Empigen BB solubilization; 30 min - Centrifugation; 10 min - IMAC purification, exchange to DDM; 2 h - Yield: 24 mg (= 1 mg/ml final) - Concentration to 12 mg/ml - SEC, concentration Cytoplasm - Crystallization at 4 o C within 7 days - Cell-free: 2.28 Å / Cellular: 2.05 Å Periplasm Boland et al (2014) CMLS

18 Systematic 3-Level Protocol Development II: Solubility = Array of expression protocols: D-CF + L-CF Type and concentration of hydrophobic compound(s), detergents, lipids, D/L-mixtures, bicelles, liposomes, nanodiscs D-CF L-CF

19 Schwarz et al (2010) Proteomics Kai et al (2013) PlosOne Throughput Screening of Expression Conditions

20 Systematic 3-Level Protocol Development III: Quality = Selection of one/few individual protocols Stability, homogeneity, activity Detergent exchange; 1 st and 2 nd detergent, additives, stabilizer Quality screening of sample arrays I II P-CF DMPC Brij58 + DMPC Brij58 DPC Triton + DMPC III

21 NMR Structure of D-CF Expressed Proteorhodopsin Reckel et al. (2011) Angew. Chem. Intl. Ed. D-CF: Digitonin:DHPC = 4:1

22 Cell-Free Expression of Multisubunit Complexes In vivo expression Cell-free expression Matthies et al (2011) J Mol Biol

23 P-CF D-CF L-CF LMPG DDM Brij35 Brij58 DMPC DMPG SDS DPC Digit T-100 Mix POPG LMPG DDM LMPG DPC Digit DHPC DDM ND-1 DMPG ND-2 Mix

24 New Production Strategy by Synthetic Biology: The Bottle Approach Classical Funnel approach New Bottle approach

25 L-CF Expression Strategies 1. High concentrations (10 mg lipid/ml) possible. 2. Designed lipid compositions. 3. Co-translational membrane protein insertion difficult. 4. Proteoliposomes precipitate after the cell-free reaction. 1. High concentrations (150 µm) possible. 2. Designed lipid compositions. 3. Co-translational membrane protein insertion more efficient. 4. Nanodisc complexes stay soluble, homogenous samples. Roos et al (2012) Mol Mem Biol

26 Synergies of CF Expression & Nanodisc Technology SugE Proteorhodopsin MraY Translocase Roos et al (2012) Biochim Biophys Acta

27 Single Molecule Force Microscopy of Membrane Proteins in Nanodiscs Bacteriorhodopsin inserts as a Trimer Zocher et al (2011) ACS Nano Roos et al (2012) BBA

28 MraY Translocase 10 TMH protein Lipid-I biosynthesis Essential for bacterial viability (Chung et al., 2013)

29 Activity (nmol/µg) Cell-Free Expression Dependent Activity of MraY translocases CF expression of functional MraY homologues Bs-MraY Ec-MraY Ma et al (2011) J Biol Chem

30 L-CF Lipid-Screening of MraY Translocases B.subtilis E.coli A.aeolicus H.pylori B.pertussis C. pneumoniae (Chung et al., 2013)

31 SPR analysis of GPCR / nanodisc complexes ETA: ET-1 = BQ-123 >> ET-3, 4-Ala-ET-1, IRL-1620 ETB: ET-1 = ET-3, 4-Ala-ET-1, IRL-1620 >> BQ-123 Kd ET-1: 1.7 ( ) Kd ET-3: 3400 (>2000) Kd ET-1: ( ) Kd ET-3: 0.2 ( ) Ligand nm IC50 (IC50 literature) ET-1 8 (0.3) Ala-4-ET (> 2000) BQ (1.4) IRL (> 500) Ligand nm IC50 (IC50 literature) ET-1 9 (0.2) Ala-4-ET-1 4 (0.33) BQ (> 1500) IRL (0.1-56) Proverbio et al (2013) Biochim Biophys Acta

32 Structures: > 1,000 globular proteins 5 membrane proteins Perspectives The Golden Age of Cell-Free Expression FEBS Letters Biochemistry: GPCRs, Transporters, Channels, Porins, Pumps, Enzymes HTP drug screening, Antibody generation System selling companies: RiNA, Cube, Promega, Invitrogen, ThermoFisher, JenaBiosciences CF generated protein selling companis: Calixar, Synthelis, Cube, Sutro 2014: Sutro Biopharma and Merck KGaA Partnership on Development of Cell-Free Produced Antibody Drug Conjugates > 200 Millions

33 Institute of Biophysical Chemistry Present: Erik Henrich, Ralf Rues, Christopher Hein, Fang Dong, Aisha Laguerre, Beate Hoffmann, Natascha Rogova Past: Davide Proverbio, Stefan Haberstock, Erika Orbán, Fredericke Junge, Sina Reckel, Christian Roos, Solmaz Sobhanifar, Daniel Schwarz, Edith Buchinger, Christian Klammt Hermann Koepsell Torsten Keller Martin Caffrey Coilin Boland Hans-Georg Sahl Tanja Schneider Bernard Mouillac