CHAPTER FIVE
An Unconventional Anaerobic Membrane Protein Production System Based on Wolinella succinogenes Michael Lafontaine, C. Roy D. Lancaster1 Department of Structural Biology, Institute of Biophysics and Center of Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 1.1 pΔfrdCAB 1.2 pFrdcat2 1.3 Construction of W. succinogenes strains 2. Methods 2.1 Materials 2.2 Preparation of growth media 2.3 Cultivation of W. succinogenes 2.4 Generation of the expression vectors 2.5 Transformation and genomic integration 2.6 Stock culture 2.7 Protein production in W. succinogenes 3. Purification of Proteins Expressed in W. succinogenes 3.1 Material 3.2 Buffers 3.3 Cell lysis procedure for the extraction of a membrane protein 3.4 Cell lysis procedure for the extraction of a periplasmic protein 3.5 Anion exchange chromatography 3.6 Size-exclusion chromatography 3.7 Determination of protein concentration 3.8 Functional characterization 3.9 Reconstitution of enzymes in proteoliposomes 3.10 Enzymic assays Acknowledgments References
Methods in Enzymology, Volume 556 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.12.026
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Abstract In cases where membrane protein production attempts in more conventional Escherichia coli-based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W. succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W. succinogenes. Subsequently, further projects using W. succinogenes as expression host are covered.
1. INTRODUCTION A prerequisite for the understanding of the mechanism of action of membrane proteins at an atomic level is the availability of accurately determined three-dimensional structures. The by far most successful technique in the determination of atomic models of membrane protein structure is X-ray crystallography ( Jaskolski & Wlodawer, 2014; Schmahl & Steurer, 2012; Wilkins, 2013). This method requires the crystallization of the membrane protein of interest (Michel, 1990; M€ uller & Lancaster, 2013; Newby et al., 2009), which in turn requires its production and purification in milligram quantities and monodisperse quality (Ostermeier & Michel, 1997). Although a number of well-established membrane protein production systems, based on bacteria (Geertsma & Poolman, 2010; Makrides, 1996; Miroux & Walker, 1996), yeast (Cereghino & Cregg, 2000; Cregg, Cereghino, Shi, & Higgins, 2000), or insect cells ( Jasti, Furukawa, Gonzales, & Gouaux, 2007), are available, (in particular heterologous) expression can fail for a variety of reasons. An alternative system for membrane proteins, where Escherichia coli-based production failed, is based on the epsilon-proteobacterium Wolinella succinogenes and is presented here. It has been demonstrated to be successful for both homologous (Herzog et al., 2012; Juhnke, Hiltscher, Nasiri, Schwalbe, & Lancaster, 2009; Lancaster, Gross, & Simon, 2001; Lancaster et al., 2000, 2005) and heterologous (Mileni et al., 2006) membrane protein production, crystallization, and membrane protein structure determination (Lancaster et al., 2000, 2001; Lancaster, Kr€ oger, Auer, & Michel, 1999; Lancaster et al., 2005; Madej,
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Nasiri, Hilgendorff, Schwalbe, & Lancaster, 2006). The system is based on earlier work by the laboratory of the late Kr€ oger (K€ ortner, Lauterbach, Tripier, Unden, & Kr€ oger, 1990; Kr€ oger et al., 2002; Lauterbach, K€ ortner, Albracht, Unden, & Kr€ oger, 1990; Simon, Gross, Ringel, Schmidt, & Kr€ oger, 1998). Of central importance is the W. succinogenes quinol:fumarate reductase (QFR) deletion mutant (ΔfrdCAB) first generated by Simon et al. (1998). In the following, the procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented for the example of QFR variants and the SdhABE complex of W. succinogenes. In addition, further projects using W. succinogenes as expression host are covered.
1.1 pΔfrdCAB Deletion of genomic frdCAB was described by Simon et al. (1998). In brief, a kanamycin deletion cassette of pUC4K was inserted in the pBR322 vector flanked by two DNA sequences corresponding to genomic regions upstream and downstream of the frdCAB operon. Ligation reactions and subsequent plasmid propagation were performed in the E. coli XL-1 blue strain. Transformation with this vector and subsequent selection with kanamycin yielded recombinant clones of W. succinogenes ΔfrdCAB mutant, where the genomic frdCAB operon was replaced with the kanamycin cassette via a double homologous recombination events. These mutants are not able to grow on fumarate but still on nitrate minimal medium.
1.2 pFrdcat2 The expression of different frdCAB variants is achieved by transforming the ΔfrdCAB mutant with the pFrdcat2 plasmid (Simon et al., 1998). This plasmid (Fig. 1A) is a derivative of the pFrd vector where the frdC2 gene and the kanamycin resistance gene (kan) are mostly deleted but it contains the chloramphenicol resistance gene (catGC) of the pDF4 vector. The sequence of this plasmid was determined by Juhnke et al. (2009) and is deposited in the EMBL nucleotide sequence database (accession no. AM909725). Ligation reactions and subsequent plasmid propagation were performed in E. coli XL-1 blue strain. Transformation with pFrdcat2 complements ΔfrdCAB as the plasmid integrates into the genome via a single recombination event between the sequence upstream of frdC in the vector and the corresponding genomic region. The resulting complemented deletion mutant, also referred
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Figure 1 Use of the pFrdcat2 vector for the generation of W. succinogenes QFR variants. (A) Construction of the W. succinogenes △frdCAB mutant. A double homologous recombination event between flanking regions present in the p△frdCAB vector and the genome of W. succinogenes leads to the replacement of the genomic frdCAB locus by a kanamycin deletion cassette (Kan). (B) Integration of the pFrdcat2 vector via a single homologous recombination event into the genome of the W. succinogenes △frdCAB mutant. The recombination event takes place between a region upstream of the frdC gene present in the pFrdcat2 vector and the genome. Simplified representation adapted from Simon et al. (1998).
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as K4, shows in terms of doubling time and fumarate reductase activity similar properties like the wild-type strain (Simon et al., 1998).
1.3 Construction of W. succinogenes strains The generation of recombinant W. succinogenes strains comprises two important steps. The first step involves the deletion of the genomic locus of a certain gene via a double homologous recombination event. In case of the QFR of W. succinogenes, the genomic frdCAB operon which encodes for the QFR subunits is replaced by a deletion cassette of the pΔfrdCAB. This deletion cassette contains a kanamycin resistance gene of pUC4K flanked by two DNA sequences corresponding to genomic regions upstream and downstream of the frdCAB operon. The flanking regions were synthesized by PCR from the pFrd and pPur adding a EcoRI and BamHI restriction site in case of the upstream fragment and BamHI and SalI for the downstream region (Simon et al., 1998). Both fragments were cloned in the pBR322 vector before inserting the kanamycin resistance gene via BamHI. Transformation of W. succinogenes with this vector and subsequent selection with kanamycin yielded recombinant clones of W. succinogenes ΔfrdCAB mutant (also referred as the deletion mutant), where the genomic frdCAB operon was replaced by the kanamycin cassette. These mutants are not able to grow on fumarate but still on nitrate minimal medium. The next step complements this mutation by integrating the pFrdcat2 vector via a single recombinant event. The pFrdcat2 vector (Fig. 1B), a modified pFrd vector, was constructed by deleting most of the kanamycin resistance gene and inserting the chloramphenicol resistance gene from pDF4a. A subsequent digestion of the vector with ClaI and SalI followed by ligation resulted in the frdCAB operon lacking the C2 open reading frame. The sequence of this plasmid is deposited in the EMBL nucleotide sequence database (accession no. AM909725). The complete pFrdcat2 plasmid is integrated into the genome via a single recombination event between the 0.7-kb fragment upstream of the frdC open reading frame in vector and genome of the deletion mutant (Fig. 2). Selection on media containing kanamycin and chloramphenicol (25 and 12.5 μg/mL, respectively) yielded the complemented deletion mutant K4 that shows wild-type properties in terms of doubling time, growth yield, and specific activity for fumarate respiration (Simon et al., 1998). For the heterologous expression of membrane protein complexes, as performed by Mileni et al. (2006), this system is still applicable. Since the QFR
Figure 2 General workflow to generate recombinant W. succinogenes strains. After generation of the expression vectors (1) and plasmid production in E. coli XL-1 blue cells (2), transformation of W. succinogenes wild-type or W. succinogenes △frdCAB mutants with the expression vectors leads to their integration into the genome (3). Selection procedure and PCR screening for genomic integration of the vector (4) yield stably transformed W. succinogenes strains that can be used for subsequent experiments (5).
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enzymes of the epsilon-proteobacterial human pathogens Helicobacter pylori (Ge et al., 2000) and Campylobacter jejuni (Weingarten, Taveirne, & Olson, 2009) have been shown to be essential for colonization of the host organism, these QFR enzymes are considered to be promising drug targets. After generating the deletion mutant as previously described (Simon et al., 1998), the cells were transformed with the pCatCj4 and pCatHpG8 plasmids. These derivatives of the pFrdcat2 vectors contain the frdCAB locus of C. jejuni and H. pylori, respectively, instead of the genuine W. succinogenes frdCAB locus. Nevertheless, the frdCAB locus is still under the control of the strong frd promoter. The derivative vectors were constructed by amplifying an frdCAB lacking fragment of pFrdcat2 and the respective frdCAB loci with primers containing a ClaI and an AvrII restriction site at their 50 ends. Transformants (W. succinogenes CjM11 and HpGM31) were able to grow on kanamycin and chloramphenicol and produced functional heterologous QFR enzymes with expression levels comparable to the homologous wild type (Mileni et al., 2006). However, this system is not only applicable to QFRs demonstrated by the work of Kern, Scheithauer, Kranz, and Simon (2010) or Juhnke et al. (2009). The latter used the genetic system to produce a nonclassical succinate:quinone oxidoreductase (SQOR) (E-type) of W. succinogenes. This enzyme, classified as an E-type SQOR (Hederstedt, 1999; Lancaster, 2002a), has not been produced yet under any tested growth condition. The SdhABE operon encodes the three distinct subunits of the enzyme which is composed of two hydrophilic subunits (SdhA and SdhB) and a membrane anchor (SdhE) which is predicted to be membrane associated via amphipathic helices than a transmembrane domain. Furthermore, the SdhA subunit contains a N-terminal 40-amino acid-long extension harboring a twin-arginine motif that predetermines the protein to be exported via the tat pathway (Palmer, Sargent, & Berks, 2005). As the cloning of the complete SdhABE operon failed, only the gene coding for sdhA subunit was amplified from genomic DNA and cloned via SacII and NotI in a pFrdcat2 fragment lacking the frdCAB operon but still containing the intact frd promoter (referred as pSdhA). Transformation of the W. succinogenes ΔfrdCAB mutant with pSdhA led to integration of the vector at the SdhABE locus putting the complete SdhABE operon under the control of the strong frd promoter. Further enzymatic activity measurements proved that a real and active protein was produced. In the same work, the compatibility of constructs with affinity tags for detection was also tested. Juhnke et al. (2009) generated pSdhAHT and
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pSdhAH1 by inserting oligonucleotide cassettes encoding a hexa-histidinetag with or without a following TEV-protease cleavage site at the start of the sdhA gene or at amino acid position 37. It turned out that care must be taken when using N-terminal affinity tags as only the AH1 variant with the hexahistidine-tag at position 37 could be detected in a Western blot with an antipenta-histidine antibody implicating that in the HT mutant, the N-terminal his-tag was cleaved during export due the tat signal peptide corresponding to amino acids 31–33 in SdhA. Furthermore, the use of a strepII-tag or a tandem strepII-tag for purification or detection of protein production was successfully demonstrated by Gross, Pisa, Sa¨nger, Lancaster, & Simon (2004) or Kern et al. (2010). However, the frd promoter is not the only useable promoter for expression of foreign or modified genes. Gross and coworkers (Gross et al., 2004; Gross, Simon, Theis, & Kroger, 1998) demonstrated the portability of the procedure of generating deletion mutants and subsequent complementation. They generated a deletion cassette harboring the kanamycin resistance gene flanked by sequences homologous to the neighboring regions of the HydABC operon. The following complementation with the pHydcat plasmid containing the HydABC operon with HydC variants as well as a chloramphenicol acetyltransferase gene yielded functional Fe/Ni hydrogenase.
2. METHODS 2.1 Materials 2.1.1 Ca/Mg solution (1000 ×) Dissolve 0.74 g CaCl2 2H2O and 5.1 g MgCl2 6H2O in 100 mL ddH2O. Autoclave and store at room temperature. 2.1.2 Cys/Glu solution (100×) Dissolve 1 g glutamate and 0.69 g L-cysteine in 100 mL ddH2O. Autoclave and store at room temperature. 2.1.3 Concentrate for nitrate media (20 ×) pH 7.5 Dissolve 121.0 g Tris, 80.8 g KNO3, 108.8 g sodium formate, 4.6 g K2HPO4 3H2O, 17.42 g K2SO4 and 11.6 g fumarate in 600 mL ddH2O. Adjust to 1 L, autoclave, and add 4 mL trace elements solution after cooling. Store at room temperature.
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2.1.4 Concentrate for fumarate media (10 ×) pH 7.9 Dissolve 60.5 g Tris, 104 g fumarate, 112 g KOH, 68 g sodium formate, 34.8 g K2HPO4, 6.6 g (NH4)2SO4, 2.7 g NH4Cl, 27.2 g sodium acetate 3H2O, and 1.5 g glutamate in 600 mL ddH2O. Adjust to 1 L and autoclave. Add 2 mL trace elements solution and store at room temperature. 2.1.5 Trace elements solution Dissolve 5.2 g Na2EDTA 2H2O, 1.5 g FeCl2 4H2O, 0.07 g ZNCl2, 0.1 g MnCl2, 0.062 g H3BO3, 190 g CoCl2 6H2O, 17 g CuCl2 2H2O, 24 g NiCl 6H2O, and 36 g NaMoO4 2H2O in 800 mL ddH2O. Adjust to 1 L and autoclave. Store at 4 °C.
2.2 Preparation of growth media W. succinogenes is grown in either minimal or rich medium with formate as electron donor and either fumarate or nitrate as electron acceptor (Bronder, Mell, Stupperich, & Kr€ oger, 1982; Lorenzen, Kr€ oger, & Unden, 1993). Rich medium is prepared by addition of 1.3% or 0.5% (m/v) brain–heart infusion (BHI, Gibco, BRL). For fumarate media, dissolve 5 g BHI in 800 mL ddH2O and add 100 mL concentrate for fumarate media (10 ) and 1 mL Ca/Mg solution (1000 ). For nitrate media, add 50 mL concentrate for nitrate media (20 ) instead and 4 mL Ca/Mg solution. Nitrate media must also be supplemented with 10 mL Cys/Glu solution (100). Adjust to 1 L with H2Odest and degas by repeated evacuation (up to 1.0 bar) and flushing with nitrogen (+0.3 bar) before autoclaving. After cooling, add 5 mL of kanamycin (5 g/L) and chloramphenicol (2.5 g/L) solution. For large-scale expression, use 10-fold higher concentrated antibiotic stock solution. 2.2.1 Soft agar Dissolve 2.6 g BHI agar in 80 mL ddH2O. Add 5 mL of concentrate for nitrate media (20) and 400 μL of Ca/Mg stock solution (1000 ). Add 100 mL with ddH2O. Degas before autoclaving and store the hot agar at 50–60 °C. Before loading the soft agar into the anaerobic chamber, add 1 mL Cys/Glu solution (100 ), 0.5 mL chloramphenicol (2.5 g/L), and 0.5 mL kanamycin (5 g/L) solution per 100 mL of agar.
2.3 Cultivation of W. succinogenes All media and solutions have to be degassed, flushed with nitrogen, and autoclaved. Therefore, the use of vacuum-safe flasks and culture tubes
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equipped with septum locks is essential to prevent breaking. For aseptically transferring anaerobic liquids and cells, pierce through the ethanol-overlaid septum with syringes with needles. Cultures are inoculated in a 1:10 ratio with a well-grown preculture. Inoculate a new culturing tube once a month.
2.4 Generation of the expression vectors Amplification of DNA for cloning or sequencing is performed using highfidelity polymerase kits, e.g., the “Expand Long Template” kit (Roche Diagnostics, Mannheim), the “BIO-X-ACT™ Long DNA Polymerase” (Bioline, Luckenwalde, Germany), or the “Phusion™ High-fidelity” PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA). For analytical PCR, the “BIOTAQ™ RED DNA Polymerase” (Bioline, Luckenwalde, Germany) is used.
2.5 Transformation and genomic integration 1. Inoculate 10 mL FoNi medium + 100 μL Cys/Glu containing 25 μg/mL kanamycin with 1 mL W. succinogenes ΔfrdCAB and let them grow over night. 2. Inoculate 10 mL FoNi medium + 100 μL Cys/Glu containing 25 μg/mL kanamycin with 1 mL of the overnight culture and incubate them for approximately 3 h at 37 °C. 3. Load the cultures into an anaerobic chamber. 4. Pour 10 mL culture into a sterile Falcon and centrifuge cells for 15 min at 5500 rpm. 5. Resuspend the cell pellet in 10 mL sucrose solution and repeat the centrifugation step above. 6. Pour off the sucrose solution and resuspend the pellet in the remaining 50 μL sucrose solution. Mix with the plasmid and transfer the reaction into a sterile electroporation cuvette. 7. Incubate for at least 5 min on ice, dry the cuvette, and put it into the cuvette holder. Prepare 1 mL prechilled medium in syringe and overlay the plug of an empty anaerobic culture tube with ethanol. Electroporate cells with 1.25 kV, 25 μF, 800 Ω. 8. Immediately add 1 mL of ice-cold medium, resuspend the cells, and transfer them with the same syringe into the prepared anaerobic tube. 9. Check the time constant Tc (Tc should be between 14 and 16 ms). 10. Unload cells from the anaerobic chamber and add 2 mL fresh medium without antibiotics after 120–150 min at 37 °C. Grow them over night.
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11. The next day use 1 mL of the overnight culture to inoculate 10 mL FoNi medium + 100 μL Cys/Glu containing 12.5 μg/mL chloramphenicol and 25 μg/mL kanamycin and grow for 2–3 days. 2.5.1 Plating 12. Use 1 mL of the well-grown culture to inoculate a fresh culture and incubate it at 37 °C up to an OD of approximately 0.3. Plate a dilution series (102, 104, 106) of the cells. Before plating cells, soft agar has to be prepared: Fill in approximately 25 mL soft agar per Petri dish and add 1 mL of the diluted cell suspension to the warm agar. Mix gently by swirling and let the agar solidify. Afterward, a prewetted Anaerocult™ pack is put to the plates into an anaerobic box that is finally unloaded. The transformation plates are incubated for 2–3 days at 37 °C. 2.5.2 Picking clones 13. Add to each well of a 24-well plate 1 mL formate/nitrate medium supplied with 1% Cys/Glu solution. Prick out one colony with a sterile 1 mL pipette tip and transfer it into the medium. Be careful not to create any air bubbles. Transfer the 24-well plate into an anaerobic box, degas, and incubate over night at 37 °C. 14. Check integration of the plasmid into the genome with whole-cell PCR with primers one of them binding to a sequence inside the plasmid and the other one binding to a sequence only present in the genome. 15. Inoculate positive clones in 10 mL nitrate medium including antibiotics (25 μg/mL kanamycin 12.5 μg/mL chloramphenicol). In the case of the complemented deletion mutant, inoculate also 10 mL fumarate medium with 1 mL of overnight culture. These cultures will grow only if a functional fumarate reductase is produced. Clones producing nonfunctional fumarate reductase enzymes or other heterologous proteins will exclusively grow on nitrate medium. 16. Verify the identity of the PCR product by sequencing. 2.5.3 Cell lysis for PCR and preparation of genomic DNA For cell lysis for sensitive applications like proof-reading PCR, pellet 50 μL of an overnight culture. Resuspend them in 50 μL 1 PCR buffer and incubate for 10 min at 95 °C. Afterward put them immediately on ice. Pellet cell debris and use 2 μL of the lysate for one PCR.
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Preparation of genomic DNA for cloning steps, Southern blotting, or sensitive PCRs is performed using the Qiagen DNeasy kit (Qiagen, Hilden, Germany). 2.5.4 Whole-cell PCR Whole-cell PCR as a fast proof of successful cloning or transformation/integration steps is performed using the BIOTAQ™ Red DNA Polymerase (Bioline, Luckenwalde, Germany).
2.6 Stock culture Verified W. succinogenes strains are stored as glycerol stocks at 20 °C as storage at 80 °C is poorly tested. Preparation of stocks is performed in an anaerobic chamber. (1) Open culture tube and pour the cells into a sterile empty 15-mL Falcon. (2) Pellet cells for 15 min at 5500 rpm and discard cell supernatant. (3) Resuspend cells in 1.5 mL fresh degassed formate/fumarate or formate/ nitrate medium and transfer the cells in a new empty, degassed, and autoclaved 10-mL culture tube. (4) Add 2 mL 87% (v/v) glycerol with a syringe and mix. (5) Incubate for 30 min at room temperature and then store the labeled tube at 20 °C.
2.7 Protein production in W. succinogenes In contrast to aerobic cultivation of E. coli, W. succinogenes cells are cultured, not in shaking flasks in incubators or fermentors, but in anaerobic culture flasks in a water bath. For a large-scale expression of the QFR, set up 60 L of formate/nitrate or formate/fumarate medium supplied with 0.5% (w/v) BHI in 10-L flasks and autoclave together with plugs with standpipes. After cooling of the medium, add antibiotics and 100 mL Cys/Glu solution per 10-L flask in case of formate/nitrate media. Addition of 2–4 drops of antifoam per 10-L flask is favorable. Finally, put on the plugs and flush the flasks with nitrogen under constant stirring via the standpipe for at least 15 min. Precultures are inoculated in two 250-mL culture flasks with 10 mL of an overnight culture and incubated at 37 °C for several hours. Inoculate the media approximately 1:100 or 1:120 (depends on the mutant) with the preculture and put the flasks in a water bath at 37 °C. Grow cells for 12–15 h in case of the QFR. Induction duration might be critical as seen in case of SdhABE, characterized as a methyl-menaquinol:fumarate reductase
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(MFR; Juhnke et al., 2009). Here, a strong decrease in enzyme activity is observed with cell densities higher than OD600 of 0.5. Harvest the cells by centrifugation for 15 min at 5000 g and flash-freeze cell pellets immediately. Store at 80 °C.
3. PURIFICATION OF PROTEINS EXPRESSED IN W. SUCCINOGENES Purification of proteins expressed in W. succinogenes is performed in an analogous manner as in E. coli. After cell disruption with pressure or enzymatic treatment, cell debris is removed with a centrifugation step of 8000 g for 20 min. Dependent on the subcellular localization of the produced protein, cytosolic, periplasmic, or membrane fractions can be prepared with different lysis and centrifugation steps. A typical procedure of the purification of a membrane protein is illustrated for the QFR as described by Unden, Hackenberg, and Kr€ oger (1980) with the modifications reported previously by Lancaster et al. (1999).
3.1 Material ¨ kta puriPeristaltic pump equipped with UV detector or FPLC (e.g., A fier, GE Healthcare) Anion exchange column (e.g., XK column packed with DEAE Sepharose Fast Flow) Size-exclusion chromatography column (e.g., HiLoad 16/600 Superdex 200 prep grade) Centrifugal filter concentrator (Centriprep™ Centifugal Filter Concentrator, Millipore) Stirred ultrafiltration cell (e.g., Model 8050 or 8200, Millipore) 1 M Dithiothreitol (DTT) 20% (w/v) Decyl-β-D-maltopyranoside (DM) 20% (w/v) Dodecyl-β-D-maltopyranoside (LM) 20% (v/v) Triton X-100
3.2 Buffers 3.2.1 10× Tris/malonate concentrate (500 mM Tris pH 7.35/20 mM malonate) Weigh in 60.57 g of Tris base and 2.08 g of malonate and dissolve in 800 mL ddH2O. Adjust at room temperature pH with acetic acid to pH 7.35. Add 1 L with ddH2O and store at room temperature.
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Table 1 Buffers for the purification of the QFR of W. succinogenes Lysis buffer AEX W1 AEX E1 AEX W2 AEX E2
SEC
50 mM Tris 50 mM Tris 50 mM Tris 50 mM Tris 50 mM Tris 20 mM pH 7.35 pH 7.35 pH 7.35 pH 7.35 pH 7.35 HEPES pH 7.3 2 mM Malonate
2 mM Malonate
2 mM Malonate
2 mM Malonate
2 mM Malonate
20 mM Malonate
1 mM DTT
1 mM DTT
1 mM DTT
1 mM DTT 1 mM DTT –
–
–
1 M NaCl
–
–
0.05% Triton X-100
0.05% Triton X-100
0.1% DM 0.1% DM 0.1% DM + 0.01% LM + 0.01% LM + 0.01% LM
0.3 M NaCl 1 mM EDTA
Composition of buffers used for the purification of W. succinogenes’ QFR is listed in Table 1. Filter-sterilize and degas all buffers. Add detergent and reducing agents directly before use.
3.3 Cell lysis procedure for the extraction of a membrane protein Resuspend cells in lysis buffer to a 10–30% (w/v) solution and homogenize. Add 1 mM PMSF, 1 mM DTT, and a few microliter of DNAse (10 mg/mL). Cell lysis is performed with an EmulsiFlex-C3 emulsifier (Avestin, Ottawa, ON, Canada) by three passes at 1400 bar. The membrane fraction is separated from the cytosolic fraction by ultracentrifugation (100,000 g, 1 h, 4 °C). Discard the supernatant and resuspend the membrane pellet in approximately 100–120 mL 1 Tris/malonate buffer supplied with 1 mM DTT and add Triton X-100 (5% of cell weight). Stir under nitrogen atmosphere for 30 min at room temperature and remove unsolubilized material by ultracentrifugation (100,000 g, 45 min).
3.4 Cell lysis procedure for the extraction of a periplasmic protein Purification of a periplasmic membrane-associated protein is illustrated in the example of the methylfumarate reductase SdhABE complex. Although
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the enzyme is membrane associated, most of specific activity is found in the soluble fractions and not in the membranes. This finding together with prediction studies points toward a periplasmic membrane-associated E-type SQOR ( Juhnke et al., 2009). For extraction of the SdhABE complex, thaw and flash-freeze 5–10 g cells in liquid nitrogen for three times and finally resuspend in 2 mL/g wet cell weight prechilled, anoxic buffer containing 50 mM Tris, pH 8.25, 1 mM malonate, and 1 mM DTT. After pelleting membranes and cell debris (36,000 g, 15 min, 4 °C), add 0.1% dodecyl-β-D-maltoside (w/v) to the supernatant and stir in a septum flask for 30 min at room temperature under continuous purging by argon gas. Pellet unsolubilized material (>200,000 g, 1 h). Purge supernatant again by argon gas and apply to anion exchange chromatography.
3.5 Anion exchange chromatography As long as there are no affinity tags fused to the produced protein, ion exchange chromatography provides a valuable method of purifying the protein of interest to homogeneity. In the case of membrane proteins like the QFR, this method is also used for detergent exchange. After solubilization, the protein containing supernatant is loaded onto a 300 mL XK column packed with DEAE Sepharose Fast Flow equilibrated in anaerobic AEX ¨ kta purifier FPLC system. After extensive washing to W1 buffer on an A remove unspecifically bound proteins, elution is performed with a linear gradient or in a stepwise fashion to a salt concentration of 1 M NaCl (AEX E1 buffer). To improve purity, the salt concentration was reduced to 0.3 M NaCl. The QFR of W. succinogenes elutes at sodium chloride concentrations of between 100 and 120 mM. After concentrating the elution fraction to approximately 20 mL in a stirred ultrafiltration cell (Model 8200, Millipore), the QFR containing elution fractions is diluted with AEX W1 to a final salt concentration of approximately 80–100 mM NaCl. Subsequently, the protein is loaded on a second DEAE column with a column volume of 100 mL equilibrated in AEX W1 buffer. The column with the bound protein is washed with several column volumes of buffer containing 0.1% (w/v) β-decyl-maltoside and 0.01% (w/v) β-dodecyl-maltoside (AEX W2 buffer) until the absorbance reaches baseline. Elution is performed by applying a linear gradient up to 300 mM NaCl (AEX E2 buffer).
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3.6 Size-exclusion chromatography After concentrating the elution fraction of the second anion exchange chromatography with a centrifugal filter concentrator to a volume of approximately 5–10 mL, the sample is injected onto a size-exclusion chromatography column with the appropriate molecular weight separation range from 10 kDa to 600 kDa (e.g., Superdex 200 16/60 or 26/60) equilibrated with SEC buffer. Monodisperse fractions are pooled, concentrated, and flash-frozen with liquid nitrogen.
3.7 Determination of protein concentration For measuring protein concentration, several popular methods can be applied like absorbance measurement at 280 nm, Bradford assay (Bradford, 1976), or BCA assay (Smith et al., 1985). The latter is used to determine protein concentration of the QFR as detergent interferes with both the absorbance measurement at 280 nm and the Bradford assay. The BCA assay is performed as described in the manufacturer’s manual (Pierce™ BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL, USA).
3.8 Functional characterization Functional characterization of proteins is performed in many ways. In this chapter, the functional characterization of the QFR of W. succinogenes is demonstrated on measuring fumarate reductase or quinol oxidation activity (Unden & Kr€ oger, 1981). This can be performed either in detergentsolubilized state (Lancaster et al., 2000) or in liposomes (Biel et al., 2002; Madej et al., 2006). The latter method was used to provide experimental evidence for the “E-pathway” (Lancaster, 2002b) for essential transmembrane proton transfer in the QFR of W. succinogenes by incorporating a “E-pathway”-defective variant into proteoliposomes (Madej et al., 2006).
3.9 Reconstitution of enzymes in proteoliposomes Reconstitution of purified enzymes of the anaerobic respiratory chain in proteoliposomes is performed according to Biel et al. (2002) and Madej et al. (2006) and comprises two steps: the preparation of sonicated liposomes and the proteoliposome reconstitution procedure. This procedure ensures a unidirectional incorporation of the enzyme into the liposomal membrane where the hydrophilic A and B subunits point outward.
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3.9.1 Material – Bio-Beads SM-2 (Bio-Rad) – Chloroform/methanol solution (2:1, v/v): Mix 6 mL chloroform with 3 mL methanol – n-Dodecyl-β-D-maltopyranoside (Anatrace) – HEPES–KCl: HEPES (5 mM) pH 7.5, KCl (100 mM) – Menaquinol (0.1 M) in ethanol (100%) – 1,2-Dipalmitoyl-sn-glycero-3 phosphocholine (phosphatidylcholine) and 2-dipalmitoyl-sn-glycero-3-phosphate (phosphatidate) (Avanti Polar Lipids, Inc.) 3.9.2 Preparation of liposomes For 5 mL of a 10 liposome stock solution: 1. Weight in 50 mg phosphatidylcholine and 5 mg phosphatidate directly into a 100-mL septum flask. 2. Dissolve lipids in 9 mL of a chloroform/methanol mixture (2:1, v/v) under stirring at room temperature. 3. Add 18.5 μL of a menaquinol stock solution (100 mM) to incorporate a quinol into the liposomal membrane. 4. Tightly close the septum flask and evaporate solvents completely by evacuating. 5. Resuspend the dry phospholipid film (layer) in 5 mL of HEPES/KCl buffer and pour the liposome suspension (11 g phospholipids/L) into a 15-mL Falcon. 6. Sonicate the liposome suspension on ice until it is completely clear (Bandelin Sonopuls equipped with microtip, 4 °C, 30 W, 40% cycle). 7. Make 1:10 dilutions with HEPES/KCl buffer in 5 mL volume in a new Falcon (1.1 g phospholipids/L). 8. Store liposomes at 20 °C until further use. 3.9.3 Preparation of proteoliposomes 9. Thaw 1 liposome suspension and sonificate on ice for 10–15 min (Bandelin Sonopuls equipped with microtip, 4 °C, 30 W, 40% cycle). 10. Transfer liposome suspension into a new 10-mL septum flask and add 0.8 g β-dodecyl-maltoside/g phospholipid and stir carefully for 3 h at room temperature. 11. Add enzyme (0.18 mg/mg phospholipids) stepwise under constant stirring for 1 h at room temperature.
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12. Remove detergent by addition of Bio-Beads SM-2 (240 mg/mL) under stirring for 1 h. 13. Transfer liposome solution into a new Falcon and remove Bio-Beads by centrifugation (5000 g, 30 s) and/or filter-sterilize (0.2 μm pore size). 14. Concentrate proteoliposomes by centrifugation at 17,000 g for 1 min. 15. Discard supernatant and resuspend pellet in 50–100 μL HEPES/ KCl buffer.
3.10 Enzymic assays 3.10.1 Material – UV–VIS Diode Array Spectrophotometer equipped with a multicell transport connected to a water bath (e.g., 8453 UV–VIS Diode Array System Agilent Technologies Inc., Santa Clara, CA, USA) – 25- or 50-μL and 1-mL Microliter syringes with needle (e.g., Hamilton Bonaduz AG, Bonaduz, Swiss) – 1-mL Quartz cuvettes (e.g., macro cells, Agilent Technologies Inc., Santa Clara, CA, USA) – Airtight plugs (Rotilabo plugs, Roth, Karlsruhe, Germany) – Potassium phosphate buffer (50 mM), pH 7.4: Filter-sterilize and degas in a septum flask – Tris buffer (50 mM), pH 8.0: Filter-sterilize and degas in a septum flask – 2,3-Dimethyl-1,4-naphthoquinone (DMN) (20 mM): Synthesized as described (Lancaster et al., 2005) – Dissolve in ethanol, protect from light, and keep on ice. Do not degas – Borohydride (20 mg/mL): Degas flask with potassium borohydride before dissolving in anaerobic ddH2O. Do not degas afterward – Benzyl viologen (0.1 M): Dissolve in anaerobic ddH2O – Sodium dithionite (50 mg/mL): Dissolve in ddH2O – Fumarate (1 M): Dissolve in H2Odest in a septum flask and degas – Fumarate (0.1 M): Dissolve in H2Odest in a septum flask and degas – Succinate (1 M): Dissolve in ddH2O in a septum flask and degas 3.10.2 Methods All enzymatic assays (Unden et al., 1980) were performed at 37 °C in 50 mM phosphate buffer, pH 7.4 in anaerobized cuvettes (path length 0.4 cm) in the presence of 150 μM DMN (Lancaster et al., 2005). Then 4–8 μL of
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the enzyme (target concentration 5–12 μg/mL) is added and the samples are incubated for 90 s prior to the measurement. 3.10.3 Measurement of the fumarate reduction activity (benzyl viologen ! fumarate assay) The fumarate reduction activity is measured using the artificial substrate benzyl viologen. It is an assay measuring the fumarate reduction activity of the two soluble subunits A and B independent of the membraneembedded C subunit. Benzyl viologen, a colorless substrate in the oxidized form, is reduced by additions of small aliquots of dithionite forming a dark violet color. As it binds artificially to the B subunit of the QFR, it serves as an electron donor for reducing fumarate. The reaction is started with the addition of fumarate and the activity is monitored by measuring the change of the absorbance at λ ¼ 546 nm (ε546 ¼ 19.5 mM1 cm1, d ¼ 0.4 cm). 1. Fill 980 μL of phosphate buffer (50 mM) pH 7.4 (or 50 mM Tris pH 8.0 in case of the SdhABE complex) in an anaerobic cuvette with a microliter syringe. 2. Add 10 μL of a benzyl viologen solution (0.1 M). 3. Reduce the compound by adding 2–5 μL of a sodium dithionite solution (40 mg/mL); absorbance at λ ¼ 546 nm should reach a value of 1.4. 4. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized. 5. Start the reaction with 10 μL of fumarate solution (1 M). 6. Calculate the initial slope of the spectrum after fumarate addition over 10 s. 3.10.4 Measuring the quinol oxidation activity (DMNH2 ! fumarate assay) The assay of quinol oxidation by fumarate is performed according to Unden and Kr€ oger (1981). The measurement is based on the quinol oxidation activity by simultaneous reduction of fumarate. In contrast to the fumarate reduction, the quinol oxidation is strictly dependent on the complete enzyme including the C subunit. As a quinol is needed, the synthetic menaquinone analogon DMN has to be pre-reduced by the addition of small aliquots of a NaBH4 solution. After starting the reaction by addition of fumarate, the reoxidation of DMNH2 is monitored spectrometrically by recording the absorbance difference at λ ¼ 270 and 290 nm (ε270–290 ¼ 15.2 mM1 cm1, d ¼ 0.4 cm). 1. Fill 975 μL of phosphate buffer (50 mM) pH 7.4 in an anaerobic cuvette with a microliter syringe.
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2. Add 10 μL of a DMN solution (20 mM). The absorbance at λ ¼ 270 nm should reach a value of 1.2. The absorbance at λ ¼ 290 nm should remain constant during the complete recording. 3. Pre-reduce the quinone by adding 2.5–5 μL of a potassium borohydride solution (20 mg/mL). The absorbance at λ ¼ 270 decreases to a value of 0.3. 4. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized. 5. Start the reaction with 10 μL of fumarate solution (0.1 M). 6. Calculate the initial slope of the difference spectra after fumarate addition over 10 s. 3.10.5 Measuring the quinone reduction activity The QFR as a SQOR is capable of reducing quinones by succinate oxidation. To measure the quinone reduction activity, the synthetic menaquinone analogon DMN is reduced by addition of succinate. The procedure is basically the same as in 3.10.4 except that the quinone is not pre-reduced by addition of potassium borohydride and succinate is used instead of fumarate. 1. Fill 970 μL of phosphate buffer (50 mM) pH 7.4 in an anaerobic cuvette with a microliter syringe. 2. Add 10 μL of a DMN solution (20 mM). The absorbance at λ ¼ 270 nm should reach a value of 1.2. The absorbance at λ ¼ 290 nm should remain constant during the complete recording. 3. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized. 4. Start the reaction with 10 μL of succinate solution (1 M). 5. Calculate the initial slope of the difference spectra after succinate addition over 10 s. 3.10.6 Calculating of the relative catalytic activity The relative catalytic activity is a useful measure for comparing protein activities. It is the ratio of the specific quinol oxidation activity and the specific fumarate reduction activity. The quotient obtained for the wild-type or reference protein is set to 100% allowing the comparison of RCA values of variants independent of preparation quality.
ACKNOWLEDGMENTS We thank all our collaborators as specified in our cited publications, in particular Hanno Juhnke, Mauro Mileni, and J€ org Simon for their contributions. Support of our research by the Deutsche Forschungsgemeinschaft (DFG, grants INST 256/275-1 FUGG and
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256/299-1 FUGG, SFB 472-Lancaster, GK 845-Lancaster, and GK 1326-Lancaster), the state of Saarland (grant LFFP 11/02), and Saarland University is gratefully acknowledged.
REFERENCES Biel, S., Simon, J., Gross, R., Ruiz, T., Ruitenberg, M., & Kr€ oger, A. (2002). Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes. European Journal of Biochemistry, 269(7), 1974–1983. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Bronder, M., Mell, H., Stupperich, E., & Kr€ oger, A. (1982). Biosynthetic pathways of Vibrio succinogenes growing with fumarate as terminal electron acceptor and sole carbon source. Archives of Microbiology, 131, 216–223. Cereghino, J. L., & Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24(1), 45–66. http://dx. doi.org/10.1111/j.1574-6976.2000.tb00532.x. Cregg, J. M., Cereghino, J. L., Shi, J. Y., & Higgins, D. R. (2000). Recombinant protein expression in Pichia pastoris. Molecular Biotechnology, 16(1), 23–52. http://dx.doi.org/ 10.1385/mb:16:1:23. Ge, Z. M., Feng, Y., Dangler, C. A., Xu, S. L., Taylor, N. S., & Fox, J. G. (2000). Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach. Microbial Pathogenesis, 29(5), 279–287. Geertsma, E. R., & Poolman, B. (2010). Production of membrane proteins in Escherichia coli and Lactococcus lactis. Methods in Molecular Biology, 601, 17–38. Gross, R., Pisa, R., Sa¨nger, M., Lancaster, C. R. D., & Simon, J. (2004). Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase. Journal of Biological Chemistry, 279(1), 274–281. Gross, R., Simon, J., Theis, F., & Kroger, A. (1998). Two membrane anchors of Wolinella succinogenes hydrogenase and their function in fumarate and polysulfide respiration. Archives of Microbiology, 170(1), 50–58. Hederstedt, L. (1999). Respiration without O2. Science, 284, 1941–1942. Herzog, E., Gu, W., Juhnke, H. D., Haas, A. H., Ma¨ntele, W., Simon, J., et al. (2012). Hydrogen-bonded networks along and bifurcation of the E-pathway in quinol:fumarate reductase. Biophysical Journal, 103(6), 1305–1314. Jaskolski, M., & Wlodawer, A. (2014). Celebrating the international year of crystallography introduction. FEBS Journal, 281(18), 3983–3984. http://dx.doi.org/10.1111/ febs.12971. Jasti, J., Furukawa, H., Gonzales, E. B., & Gouaux, E. (2007). Structure of acid-sensing ion channel 1 at 1.9A resolution and low pH. Nature, 449(7160), 316–323. Juhnke, H. D., Hiltscher, H., Nasiri, H. R., Schwalbe, H., & Lancaster, C. R. D. (2009). Production, characterization and determination of the real catalytic properties of the putative ‘succinate dehydrogenase’ from Wolinella succinogenes. Molecular Microbiology, 71(5), 1088–1101. Kern, M., Scheithauer, J., Kranz, R. G., & Simon, J. (2010). Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases. Microbiology (Reading, England), 156, 3773–3781. K€ ortner, C., Lauterbach, F., Tripier, D., Unden, G., & Kr€ oger, A. (1990). Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b. Molecular Microbiology, 4, 855–860.
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Kr€ oger, A., Biel, S., Simon, J., Gross, R., Unden, G., & Lancaster, C. R. D. (2002). Fumarate respiration of Wolinella succinogenes: Enzymology, energetics and coupling mechanism. Biochimica et Biophysica Acta, 1553(1–2), 23–38. Lancaster, C. R. D. (2002a). Succinate:quinone oxidoreductases: An overview. Biochimica et Biophysica Acta, 1553, 1–6. Lancaster, C. R. D. (2002b). Wolinella succinogenes quinol:fumarate reductase—2.2-A resolution crystal structure and the E-pathway hypothesis of coupled transmembrane proton and electron transfer. Biochimica et Biophysica Acta, 1565(2), 215–231. Lancaster, C. R. D., Gross, R., Haas, A., Ritter, M., Ma¨ntele, W., Simon, J., et al. (2000). Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase. Proceedings of the National Academy of Sciences of the United States of America, 97, 13051–13056. Lancaster, C. R. D., Gross, R., & Simon, J. (2001). A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction. European Journal of Biochemistry, 268, 1820–1827. Lancaster, C. R. D., Kr€ oger, A., Auer, M., & Michel, H. (1999). Structure of fumarate reductase from Wolinella succinogenes at 2.2 angstrom resolution. Nature, 402, 377–385. Lancaster, C. R. D., Sauer, U. S., Gross, R., Haas, A. H., Graf, J., Schwalbe, H., et al. (2005). Experimental support for the “E pathway hypothesis” of coupled transmembrane e() and H + transfer in dihemic quinol:fumarate reductase. Proceedings of the National Academy of Sciences of the United States of America, 102, 18860–18865. Lauterbach, F., K€ ortner, C., Albracht, S. P. J., Unden, G., & Kr€ oger, A. (1990). The fumarate reductase operon of Wolinella succinogenes. Sequence and expression of the frdA and frdB genes. Archives of Microbiology, 154, 386–393. Lorenzen, J. P., Kr€ oger, A., & Unden, G. (1993). Regulation of anaerobic respiratory pathways in Wolinella succinogenes by the presence of electron acceptors. Archives of Microbiology, 159, 477–483. Madej, M. G., Nasiri, H. R., Hilgendorff, N. S., Schwalbe, H., & Lancaster, C. R. D. (2006). Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex. EMBO Journal, 25, 4963–4970. Makrides, S. C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Reviews, 60(3), 512–538. Michel, H. (Ed.), (1990). Crystallization of membrane proteins. Boca Raton: CRC Press. Mileni, M., MacMillan, F., Tziatzios, C., Zwicker, K., Haas, A. H., Ma¨ntele, W., et al. (2006). Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni. Biochemical Journal, 395, 191–201. Miroux, B., & Walker, J. E. (1996). Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology, 260(3), 289–298. http://dx.doi.org/10.1006/ jmbi.1996.0399. M€ uller, F. G., & Lancaster, C. R. D. (2013). Crystallization of membrane proteins. Methods in Molecular Biology, 1033, 67–83. Newby, Z. E. R., O’Connell, J. D., Gruswitz, F., Hays, F. A., Harries, W. E. C., Harwood, I. M., et al. (2009). A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nature Protocols, 4(5), 619–637. Ostermeier, C., & Michel, H. (1997). Crystallization of membrane proteins. Current Opinion in Structural Biology, 7, 697–701. Palmer, T., Sargent, F., & Berks, B. C. (2005). Export of complex cofactor-containing proteins by the bacterial Tat pathway. Trends in Microbiology, 13, 175–180.
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Schmahl, W. W., & Steurer, W. (2012). Laue centennial. Acta Crystallographica. Section A, Foundations of Crystallography, 68(1), 1–2. http://dx.doi.org/10.1107/ S0108767311041353. Simon, J., Gross, R., Ringel, M., Schmidt, E., & Kr€ oger, A. (1998). Deletion and sitedirected mutagenesis of the Wolinella succinogenes fumarate reductase operon. European Journal of Biochemistry, 251, 418–426. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., et al. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150, 76–85. Unden, G., Hackenberg, H., & Kr€ oger, A. (1980). Isolation and functional aspects of the fumarate reductase involved in the phosphorylative electron transport of Vibrio succinogenes. Biochimica et Biophysica Acta, 591, 275–288. Unden, G., & Kr€ oger, A. (1981). The function fo the subunits of the fumarate reductase complex of Vibrio succinogenes. European Journal of Biochemistry, 120, 577–584. Weingarten, R. A., Taveirne, M. E., & Olson, J. W. (2009). The dual-functioning fumarate reductase is the sole succinate: Quinone reductase in Campylobacter jejuni and is required for full host colonization. Journal of Bacteriology, 191(16), 5293–5300. Wilkins, S. (2013). Celebrating 100 years of X-ray crystallography. Acta Crystallographica. Section A, 69(1), 1–4. http://dx.doi.org/10.1107/S0108767312048490.
An Unconventional Anaerobic Membrane Protein Production System Based on Wolinella succinogenes Michael Lafontaine, C. Roy D. Lancaster1 Department of Structural Biology, Institute of Biophysics and Center of Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 1.1 pΔfrdCAB 1.2 pFrdcat2 1.3 Construction of W. succinogenes strains 2. Methods 2.1 Materials 2.2 Preparation of growth media 2.3 Cultivation of W. succinogenes 2.4 Generation of the expression vectors 2.5 Transformation and genomic integration 2.6 Stock culture 2.7 Protein production in W. succinogenes 3. Purification of Proteins Expressed in W. succinogenes 3.1 Material 3.2 Buffers 3.3 Cell lysis procedure for the extraction of a membrane protein 3.4 Cell lysis procedure for the extraction of a periplasmic protein 3.5 Anion exchange chromatography 3.6 Size-exclusion chromatography 3.7 Determination of protein concentration 3.8 Functional characterization 3.9 Reconstitution of enzymes in proteoliposomes 3.10 Enzymic assays Acknowledgments References
Methods in Enzymology, Volume 556 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.12.026
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Abstract In cases where membrane protein production attempts in more conventional Escherichia coli-based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W. succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W. succinogenes. Subsequently, further projects using W. succinogenes as expression host are covered.
1. INTRODUCTION A prerequisite for the understanding of the mechanism of action of membrane proteins at an atomic level is the availability of accurately determined three-dimensional structures. The by far most successful technique in the determination of atomic models of membrane protein structure is X-ray crystallography ( Jaskolski & Wlodawer, 2014; Schmahl & Steurer, 2012; Wilkins, 2013). This method requires the crystallization of the membrane protein of interest (Michel, 1990; M€ uller & Lancaster, 2013; Newby et al., 2009), which in turn requires its production and purification in milligram quantities and monodisperse quality (Ostermeier & Michel, 1997). Although a number of well-established membrane protein production systems, based on bacteria (Geertsma & Poolman, 2010; Makrides, 1996; Miroux & Walker, 1996), yeast (Cereghino & Cregg, 2000; Cregg, Cereghino, Shi, & Higgins, 2000), or insect cells ( Jasti, Furukawa, Gonzales, & Gouaux, 2007), are available, (in particular heterologous) expression can fail for a variety of reasons. An alternative system for membrane proteins, where Escherichia coli-based production failed, is based on the epsilon-proteobacterium Wolinella succinogenes and is presented here. It has been demonstrated to be successful for both homologous (Herzog et al., 2012; Juhnke, Hiltscher, Nasiri, Schwalbe, & Lancaster, 2009; Lancaster, Gross, & Simon, 2001; Lancaster et al., 2000, 2005) and heterologous (Mileni et al., 2006) membrane protein production, crystallization, and membrane protein structure determination (Lancaster et al., 2000, 2001; Lancaster, Kr€ oger, Auer, & Michel, 1999; Lancaster et al., 2005; Madej,
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Nasiri, Hilgendorff, Schwalbe, & Lancaster, 2006). The system is based on earlier work by the laboratory of the late Kr€ oger (K€ ortner, Lauterbach, Tripier, Unden, & Kr€ oger, 1990; Kr€ oger et al., 2002; Lauterbach, K€ ortner, Albracht, Unden, & Kr€ oger, 1990; Simon, Gross, Ringel, Schmidt, & Kr€ oger, 1998). Of central importance is the W. succinogenes quinol:fumarate reductase (QFR) deletion mutant (ΔfrdCAB) first generated by Simon et al. (1998). In the following, the procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented for the example of QFR variants and the SdhABE complex of W. succinogenes. In addition, further projects using W. succinogenes as expression host are covered.
1.1 pΔfrdCAB Deletion of genomic frdCAB was described by Simon et al. (1998). In brief, a kanamycin deletion cassette of pUC4K was inserted in the pBR322 vector flanked by two DNA sequences corresponding to genomic regions upstream and downstream of the frdCAB operon. Ligation reactions and subsequent plasmid propagation were performed in the E. coli XL-1 blue strain. Transformation with this vector and subsequent selection with kanamycin yielded recombinant clones of W. succinogenes ΔfrdCAB mutant, where the genomic frdCAB operon was replaced with the kanamycin cassette via a double homologous recombination events. These mutants are not able to grow on fumarate but still on nitrate minimal medium.
1.2 pFrdcat2 The expression of different frdCAB variants is achieved by transforming the ΔfrdCAB mutant with the pFrdcat2 plasmid (Simon et al., 1998). This plasmid (Fig. 1A) is a derivative of the pFrd vector where the frdC2 gene and the kanamycin resistance gene (kan) are mostly deleted but it contains the chloramphenicol resistance gene (catGC) of the pDF4 vector. The sequence of this plasmid was determined by Juhnke et al. (2009) and is deposited in the EMBL nucleotide sequence database (accession no. AM909725). Ligation reactions and subsequent plasmid propagation were performed in E. coli XL-1 blue strain. Transformation with pFrdcat2 complements ΔfrdCAB as the plasmid integrates into the genome via a single recombination event between the sequence upstream of frdC in the vector and the corresponding genomic region. The resulting complemented deletion mutant, also referred
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Figure 1 Use of the pFrdcat2 vector for the generation of W. succinogenes QFR variants. (A) Construction of the W. succinogenes △frdCAB mutant. A double homologous recombination event between flanking regions present in the p△frdCAB vector and the genome of W. succinogenes leads to the replacement of the genomic frdCAB locus by a kanamycin deletion cassette (Kan). (B) Integration of the pFrdcat2 vector via a single homologous recombination event into the genome of the W. succinogenes △frdCAB mutant. The recombination event takes place between a region upstream of the frdC gene present in the pFrdcat2 vector and the genome. Simplified representation adapted from Simon et al. (1998).
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as K4, shows in terms of doubling time and fumarate reductase activity similar properties like the wild-type strain (Simon et al., 1998).
1.3 Construction of W. succinogenes strains The generation of recombinant W. succinogenes strains comprises two important steps. The first step involves the deletion of the genomic locus of a certain gene via a double homologous recombination event. In case of the QFR of W. succinogenes, the genomic frdCAB operon which encodes for the QFR subunits is replaced by a deletion cassette of the pΔfrdCAB. This deletion cassette contains a kanamycin resistance gene of pUC4K flanked by two DNA sequences corresponding to genomic regions upstream and downstream of the frdCAB operon. The flanking regions were synthesized by PCR from the pFrd and pPur adding a EcoRI and BamHI restriction site in case of the upstream fragment and BamHI and SalI for the downstream region (Simon et al., 1998). Both fragments were cloned in the pBR322 vector before inserting the kanamycin resistance gene via BamHI. Transformation of W. succinogenes with this vector and subsequent selection with kanamycin yielded recombinant clones of W. succinogenes ΔfrdCAB mutant (also referred as the deletion mutant), where the genomic frdCAB operon was replaced by the kanamycin cassette. These mutants are not able to grow on fumarate but still on nitrate minimal medium. The next step complements this mutation by integrating the pFrdcat2 vector via a single recombinant event. The pFrdcat2 vector (Fig. 1B), a modified pFrd vector, was constructed by deleting most of the kanamycin resistance gene and inserting the chloramphenicol resistance gene from pDF4a. A subsequent digestion of the vector with ClaI and SalI followed by ligation resulted in the frdCAB operon lacking the C2 open reading frame. The sequence of this plasmid is deposited in the EMBL nucleotide sequence database (accession no. AM909725). The complete pFrdcat2 plasmid is integrated into the genome via a single recombination event between the 0.7-kb fragment upstream of the frdC open reading frame in vector and genome of the deletion mutant (Fig. 2). Selection on media containing kanamycin and chloramphenicol (25 and 12.5 μg/mL, respectively) yielded the complemented deletion mutant K4 that shows wild-type properties in terms of doubling time, growth yield, and specific activity for fumarate respiration (Simon et al., 1998). For the heterologous expression of membrane protein complexes, as performed by Mileni et al. (2006), this system is still applicable. Since the QFR
Figure 2 General workflow to generate recombinant W. succinogenes strains. After generation of the expression vectors (1) and plasmid production in E. coli XL-1 blue cells (2), transformation of W. succinogenes wild-type or W. succinogenes △frdCAB mutants with the expression vectors leads to their integration into the genome (3). Selection procedure and PCR screening for genomic integration of the vector (4) yield stably transformed W. succinogenes strains that can be used for subsequent experiments (5).
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enzymes of the epsilon-proteobacterial human pathogens Helicobacter pylori (Ge et al., 2000) and Campylobacter jejuni (Weingarten, Taveirne, & Olson, 2009) have been shown to be essential for colonization of the host organism, these QFR enzymes are considered to be promising drug targets. After generating the deletion mutant as previously described (Simon et al., 1998), the cells were transformed with the pCatCj4 and pCatHpG8 plasmids. These derivatives of the pFrdcat2 vectors contain the frdCAB locus of C. jejuni and H. pylori, respectively, instead of the genuine W. succinogenes frdCAB locus. Nevertheless, the frdCAB locus is still under the control of the strong frd promoter. The derivative vectors were constructed by amplifying an frdCAB lacking fragment of pFrdcat2 and the respective frdCAB loci with primers containing a ClaI and an AvrII restriction site at their 50 ends. Transformants (W. succinogenes CjM11 and HpGM31) were able to grow on kanamycin and chloramphenicol and produced functional heterologous QFR enzymes with expression levels comparable to the homologous wild type (Mileni et al., 2006). However, this system is not only applicable to QFRs demonstrated by the work of Kern, Scheithauer, Kranz, and Simon (2010) or Juhnke et al. (2009). The latter used the genetic system to produce a nonclassical succinate:quinone oxidoreductase (SQOR) (E-type) of W. succinogenes. This enzyme, classified as an E-type SQOR (Hederstedt, 1999; Lancaster, 2002a), has not been produced yet under any tested growth condition. The SdhABE operon encodes the three distinct subunits of the enzyme which is composed of two hydrophilic subunits (SdhA and SdhB) and a membrane anchor (SdhE) which is predicted to be membrane associated via amphipathic helices than a transmembrane domain. Furthermore, the SdhA subunit contains a N-terminal 40-amino acid-long extension harboring a twin-arginine motif that predetermines the protein to be exported via the tat pathway (Palmer, Sargent, & Berks, 2005). As the cloning of the complete SdhABE operon failed, only the gene coding for sdhA subunit was amplified from genomic DNA and cloned via SacII and NotI in a pFrdcat2 fragment lacking the frdCAB operon but still containing the intact frd promoter (referred as pSdhA). Transformation of the W. succinogenes ΔfrdCAB mutant with pSdhA led to integration of the vector at the SdhABE locus putting the complete SdhABE operon under the control of the strong frd promoter. Further enzymatic activity measurements proved that a real and active protein was produced. In the same work, the compatibility of constructs with affinity tags for detection was also tested. Juhnke et al. (2009) generated pSdhAHT and
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pSdhAH1 by inserting oligonucleotide cassettes encoding a hexa-histidinetag with or without a following TEV-protease cleavage site at the start of the sdhA gene or at amino acid position 37. It turned out that care must be taken when using N-terminal affinity tags as only the AH1 variant with the hexahistidine-tag at position 37 could be detected in a Western blot with an antipenta-histidine antibody implicating that in the HT mutant, the N-terminal his-tag was cleaved during export due the tat signal peptide corresponding to amino acids 31–33 in SdhA. Furthermore, the use of a strepII-tag or a tandem strepII-tag for purification or detection of protein production was successfully demonstrated by Gross, Pisa, Sa¨nger, Lancaster, & Simon (2004) or Kern et al. (2010). However, the frd promoter is not the only useable promoter for expression of foreign or modified genes. Gross and coworkers (Gross et al., 2004; Gross, Simon, Theis, & Kroger, 1998) demonstrated the portability of the procedure of generating deletion mutants and subsequent complementation. They generated a deletion cassette harboring the kanamycin resistance gene flanked by sequences homologous to the neighboring regions of the HydABC operon. The following complementation with the pHydcat plasmid containing the HydABC operon with HydC variants as well as a chloramphenicol acetyltransferase gene yielded functional Fe/Ni hydrogenase.
2. METHODS 2.1 Materials 2.1.1 Ca/Mg solution (1000 ×) Dissolve 0.74 g CaCl2 2H2O and 5.1 g MgCl2 6H2O in 100 mL ddH2O. Autoclave and store at room temperature. 2.1.2 Cys/Glu solution (100×) Dissolve 1 g glutamate and 0.69 g L-cysteine in 100 mL ddH2O. Autoclave and store at room temperature. 2.1.3 Concentrate for nitrate media (20 ×) pH 7.5 Dissolve 121.0 g Tris, 80.8 g KNO3, 108.8 g sodium formate, 4.6 g K2HPO4 3H2O, 17.42 g K2SO4 and 11.6 g fumarate in 600 mL ddH2O. Adjust to 1 L, autoclave, and add 4 mL trace elements solution after cooling. Store at room temperature.
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2.1.4 Concentrate for fumarate media (10 ×) pH 7.9 Dissolve 60.5 g Tris, 104 g fumarate, 112 g KOH, 68 g sodium formate, 34.8 g K2HPO4, 6.6 g (NH4)2SO4, 2.7 g NH4Cl, 27.2 g sodium acetate 3H2O, and 1.5 g glutamate in 600 mL ddH2O. Adjust to 1 L and autoclave. Add 2 mL trace elements solution and store at room temperature. 2.1.5 Trace elements solution Dissolve 5.2 g Na2EDTA 2H2O, 1.5 g FeCl2 4H2O, 0.07 g ZNCl2, 0.1 g MnCl2, 0.062 g H3BO3, 190 g CoCl2 6H2O, 17 g CuCl2 2H2O, 24 g NiCl 6H2O, and 36 g NaMoO4 2H2O in 800 mL ddH2O. Adjust to 1 L and autoclave. Store at 4 °C.
2.2 Preparation of growth media W. succinogenes is grown in either minimal or rich medium with formate as electron donor and either fumarate or nitrate as electron acceptor (Bronder, Mell, Stupperich, & Kr€ oger, 1982; Lorenzen, Kr€ oger, & Unden, 1993). Rich medium is prepared by addition of 1.3% or 0.5% (m/v) brain–heart infusion (BHI, Gibco, BRL). For fumarate media, dissolve 5 g BHI in 800 mL ddH2O and add 100 mL concentrate for fumarate media (10 ) and 1 mL Ca/Mg solution (1000 ). For nitrate media, add 50 mL concentrate for nitrate media (20 ) instead and 4 mL Ca/Mg solution. Nitrate media must also be supplemented with 10 mL Cys/Glu solution (100). Adjust to 1 L with H2Odest and degas by repeated evacuation (up to 1.0 bar) and flushing with nitrogen (+0.3 bar) before autoclaving. After cooling, add 5 mL of kanamycin (5 g/L) and chloramphenicol (2.5 g/L) solution. For large-scale expression, use 10-fold higher concentrated antibiotic stock solution. 2.2.1 Soft agar Dissolve 2.6 g BHI agar in 80 mL ddH2O. Add 5 mL of concentrate for nitrate media (20) and 400 μL of Ca/Mg stock solution (1000 ). Add 100 mL with ddH2O. Degas before autoclaving and store the hot agar at 50–60 °C. Before loading the soft agar into the anaerobic chamber, add 1 mL Cys/Glu solution (100 ), 0.5 mL chloramphenicol (2.5 g/L), and 0.5 mL kanamycin (5 g/L) solution per 100 mL of agar.
2.3 Cultivation of W. succinogenes All media and solutions have to be degassed, flushed with nitrogen, and autoclaved. Therefore, the use of vacuum-safe flasks and culture tubes
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equipped with septum locks is essential to prevent breaking. For aseptically transferring anaerobic liquids and cells, pierce through the ethanol-overlaid septum with syringes with needles. Cultures are inoculated in a 1:10 ratio with a well-grown preculture. Inoculate a new culturing tube once a month.
2.4 Generation of the expression vectors Amplification of DNA for cloning or sequencing is performed using highfidelity polymerase kits, e.g., the “Expand Long Template” kit (Roche Diagnostics, Mannheim), the “BIO-X-ACT™ Long DNA Polymerase” (Bioline, Luckenwalde, Germany), or the “Phusion™ High-fidelity” PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA). For analytical PCR, the “BIOTAQ™ RED DNA Polymerase” (Bioline, Luckenwalde, Germany) is used.
2.5 Transformation and genomic integration 1. Inoculate 10 mL FoNi medium + 100 μL Cys/Glu containing 25 μg/mL kanamycin with 1 mL W. succinogenes ΔfrdCAB and let them grow over night. 2. Inoculate 10 mL FoNi medium + 100 μL Cys/Glu containing 25 μg/mL kanamycin with 1 mL of the overnight culture and incubate them for approximately 3 h at 37 °C. 3. Load the cultures into an anaerobic chamber. 4. Pour 10 mL culture into a sterile Falcon and centrifuge cells for 15 min at 5500 rpm. 5. Resuspend the cell pellet in 10 mL sucrose solution and repeat the centrifugation step above. 6. Pour off the sucrose solution and resuspend the pellet in the remaining 50 μL sucrose solution. Mix with the plasmid and transfer the reaction into a sterile electroporation cuvette. 7. Incubate for at least 5 min on ice, dry the cuvette, and put it into the cuvette holder. Prepare 1 mL prechilled medium in syringe and overlay the plug of an empty anaerobic culture tube with ethanol. Electroporate cells with 1.25 kV, 25 μF, 800 Ω. 8. Immediately add 1 mL of ice-cold medium, resuspend the cells, and transfer them with the same syringe into the prepared anaerobic tube. 9. Check the time constant Tc (Tc should be between 14 and 16 ms). 10. Unload cells from the anaerobic chamber and add 2 mL fresh medium without antibiotics after 120–150 min at 37 °C. Grow them over night.
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11. The next day use 1 mL of the overnight culture to inoculate 10 mL FoNi medium + 100 μL Cys/Glu containing 12.5 μg/mL chloramphenicol and 25 μg/mL kanamycin and grow for 2–3 days. 2.5.1 Plating 12. Use 1 mL of the well-grown culture to inoculate a fresh culture and incubate it at 37 °C up to an OD of approximately 0.3. Plate a dilution series (102, 104, 106) of the cells. Before plating cells, soft agar has to be prepared: Fill in approximately 25 mL soft agar per Petri dish and add 1 mL of the diluted cell suspension to the warm agar. Mix gently by swirling and let the agar solidify. Afterward, a prewetted Anaerocult™ pack is put to the plates into an anaerobic box that is finally unloaded. The transformation plates are incubated for 2–3 days at 37 °C. 2.5.2 Picking clones 13. Add to each well of a 24-well plate 1 mL formate/nitrate medium supplied with 1% Cys/Glu solution. Prick out one colony with a sterile 1 mL pipette tip and transfer it into the medium. Be careful not to create any air bubbles. Transfer the 24-well plate into an anaerobic box, degas, and incubate over night at 37 °C. 14. Check integration of the plasmid into the genome with whole-cell PCR with primers one of them binding to a sequence inside the plasmid and the other one binding to a sequence only present in the genome. 15. Inoculate positive clones in 10 mL nitrate medium including antibiotics (25 μg/mL kanamycin 12.5 μg/mL chloramphenicol). In the case of the complemented deletion mutant, inoculate also 10 mL fumarate medium with 1 mL of overnight culture. These cultures will grow only if a functional fumarate reductase is produced. Clones producing nonfunctional fumarate reductase enzymes or other heterologous proteins will exclusively grow on nitrate medium. 16. Verify the identity of the PCR product by sequencing. 2.5.3 Cell lysis for PCR and preparation of genomic DNA For cell lysis for sensitive applications like proof-reading PCR, pellet 50 μL of an overnight culture. Resuspend them in 50 μL 1 PCR buffer and incubate for 10 min at 95 °C. Afterward put them immediately on ice. Pellet cell debris and use 2 μL of the lysate for one PCR.
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Preparation of genomic DNA for cloning steps, Southern blotting, or sensitive PCRs is performed using the Qiagen DNeasy kit (Qiagen, Hilden, Germany). 2.5.4 Whole-cell PCR Whole-cell PCR as a fast proof of successful cloning or transformation/integration steps is performed using the BIOTAQ™ Red DNA Polymerase (Bioline, Luckenwalde, Germany).
2.6 Stock culture Verified W. succinogenes strains are stored as glycerol stocks at 20 °C as storage at 80 °C is poorly tested. Preparation of stocks is performed in an anaerobic chamber. (1) Open culture tube and pour the cells into a sterile empty 15-mL Falcon. (2) Pellet cells for 15 min at 5500 rpm and discard cell supernatant. (3) Resuspend cells in 1.5 mL fresh degassed formate/fumarate or formate/ nitrate medium and transfer the cells in a new empty, degassed, and autoclaved 10-mL culture tube. (4) Add 2 mL 87% (v/v) glycerol with a syringe and mix. (5) Incubate for 30 min at room temperature and then store the labeled tube at 20 °C.
2.7 Protein production in W. succinogenes In contrast to aerobic cultivation of E. coli, W. succinogenes cells are cultured, not in shaking flasks in incubators or fermentors, but in anaerobic culture flasks in a water bath. For a large-scale expression of the QFR, set up 60 L of formate/nitrate or formate/fumarate medium supplied with 0.5% (w/v) BHI in 10-L flasks and autoclave together with plugs with standpipes. After cooling of the medium, add antibiotics and 100 mL Cys/Glu solution per 10-L flask in case of formate/nitrate media. Addition of 2–4 drops of antifoam per 10-L flask is favorable. Finally, put on the plugs and flush the flasks with nitrogen under constant stirring via the standpipe for at least 15 min. Precultures are inoculated in two 250-mL culture flasks with 10 mL of an overnight culture and incubated at 37 °C for several hours. Inoculate the media approximately 1:100 or 1:120 (depends on the mutant) with the preculture and put the flasks in a water bath at 37 °C. Grow cells for 12–15 h in case of the QFR. Induction duration might be critical as seen in case of SdhABE, characterized as a methyl-menaquinol:fumarate reductase
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(MFR; Juhnke et al., 2009). Here, a strong decrease in enzyme activity is observed with cell densities higher than OD600 of 0.5. Harvest the cells by centrifugation for 15 min at 5000 g and flash-freeze cell pellets immediately. Store at 80 °C.
3. PURIFICATION OF PROTEINS EXPRESSED IN W. SUCCINOGENES Purification of proteins expressed in W. succinogenes is performed in an analogous manner as in E. coli. After cell disruption with pressure or enzymatic treatment, cell debris is removed with a centrifugation step of 8000 g for 20 min. Dependent on the subcellular localization of the produced protein, cytosolic, periplasmic, or membrane fractions can be prepared with different lysis and centrifugation steps. A typical procedure of the purification of a membrane protein is illustrated for the QFR as described by Unden, Hackenberg, and Kr€ oger (1980) with the modifications reported previously by Lancaster et al. (1999).
3.1 Material ¨ kta puriPeristaltic pump equipped with UV detector or FPLC (e.g., A fier, GE Healthcare) Anion exchange column (e.g., XK column packed with DEAE Sepharose Fast Flow) Size-exclusion chromatography column (e.g., HiLoad 16/600 Superdex 200 prep grade) Centrifugal filter concentrator (Centriprep™ Centifugal Filter Concentrator, Millipore) Stirred ultrafiltration cell (e.g., Model 8050 or 8200, Millipore) 1 M Dithiothreitol (DTT) 20% (w/v) Decyl-β-D-maltopyranoside (DM) 20% (w/v) Dodecyl-β-D-maltopyranoside (LM) 20% (v/v) Triton X-100
3.2 Buffers 3.2.1 10× Tris/malonate concentrate (500 mM Tris pH 7.35/20 mM malonate) Weigh in 60.57 g of Tris base and 2.08 g of malonate and dissolve in 800 mL ddH2O. Adjust at room temperature pH with acetic acid to pH 7.35. Add 1 L with ddH2O and store at room temperature.
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Table 1 Buffers for the purification of the QFR of W. succinogenes Lysis buffer AEX W1 AEX E1 AEX W2 AEX E2
SEC
50 mM Tris 50 mM Tris 50 mM Tris 50 mM Tris 50 mM Tris 20 mM pH 7.35 pH 7.35 pH 7.35 pH 7.35 pH 7.35 HEPES pH 7.3 2 mM Malonate
2 mM Malonate
2 mM Malonate
2 mM Malonate
2 mM Malonate
20 mM Malonate
1 mM DTT
1 mM DTT
1 mM DTT
1 mM DTT 1 mM DTT –
–
–
1 M NaCl
–
–
0.05% Triton X-100
0.05% Triton X-100
0.1% DM 0.1% DM 0.1% DM + 0.01% LM + 0.01% LM + 0.01% LM
0.3 M NaCl 1 mM EDTA
Composition of buffers used for the purification of W. succinogenes’ QFR is listed in Table 1. Filter-sterilize and degas all buffers. Add detergent and reducing agents directly before use.
3.3 Cell lysis procedure for the extraction of a membrane protein Resuspend cells in lysis buffer to a 10–30% (w/v) solution and homogenize. Add 1 mM PMSF, 1 mM DTT, and a few microliter of DNAse (10 mg/mL). Cell lysis is performed with an EmulsiFlex-C3 emulsifier (Avestin, Ottawa, ON, Canada) by three passes at 1400 bar. The membrane fraction is separated from the cytosolic fraction by ultracentrifugation (100,000 g, 1 h, 4 °C). Discard the supernatant and resuspend the membrane pellet in approximately 100–120 mL 1 Tris/malonate buffer supplied with 1 mM DTT and add Triton X-100 (5% of cell weight). Stir under nitrogen atmosphere for 30 min at room temperature and remove unsolubilized material by ultracentrifugation (100,000 g, 45 min).
3.4 Cell lysis procedure for the extraction of a periplasmic protein Purification of a periplasmic membrane-associated protein is illustrated in the example of the methylfumarate reductase SdhABE complex. Although
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the enzyme is membrane associated, most of specific activity is found in the soluble fractions and not in the membranes. This finding together with prediction studies points toward a periplasmic membrane-associated E-type SQOR ( Juhnke et al., 2009). For extraction of the SdhABE complex, thaw and flash-freeze 5–10 g cells in liquid nitrogen for three times and finally resuspend in 2 mL/g wet cell weight prechilled, anoxic buffer containing 50 mM Tris, pH 8.25, 1 mM malonate, and 1 mM DTT. After pelleting membranes and cell debris (36,000 g, 15 min, 4 °C), add 0.1% dodecyl-β-D-maltoside (w/v) to the supernatant and stir in a septum flask for 30 min at room temperature under continuous purging by argon gas. Pellet unsolubilized material (>200,000 g, 1 h). Purge supernatant again by argon gas and apply to anion exchange chromatography.
3.5 Anion exchange chromatography As long as there are no affinity tags fused to the produced protein, ion exchange chromatography provides a valuable method of purifying the protein of interest to homogeneity. In the case of membrane proteins like the QFR, this method is also used for detergent exchange. After solubilization, the protein containing supernatant is loaded onto a 300 mL XK column packed with DEAE Sepharose Fast Flow equilibrated in anaerobic AEX ¨ kta purifier FPLC system. After extensive washing to W1 buffer on an A remove unspecifically bound proteins, elution is performed with a linear gradient or in a stepwise fashion to a salt concentration of 1 M NaCl (AEX E1 buffer). To improve purity, the salt concentration was reduced to 0.3 M NaCl. The QFR of W. succinogenes elutes at sodium chloride concentrations of between 100 and 120 mM. After concentrating the elution fraction to approximately 20 mL in a stirred ultrafiltration cell (Model 8200, Millipore), the QFR containing elution fractions is diluted with AEX W1 to a final salt concentration of approximately 80–100 mM NaCl. Subsequently, the protein is loaded on a second DEAE column with a column volume of 100 mL equilibrated in AEX W1 buffer. The column with the bound protein is washed with several column volumes of buffer containing 0.1% (w/v) β-decyl-maltoside and 0.01% (w/v) β-dodecyl-maltoside (AEX W2 buffer) until the absorbance reaches baseline. Elution is performed by applying a linear gradient up to 300 mM NaCl (AEX E2 buffer).
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3.6 Size-exclusion chromatography After concentrating the elution fraction of the second anion exchange chromatography with a centrifugal filter concentrator to a volume of approximately 5–10 mL, the sample is injected onto a size-exclusion chromatography column with the appropriate molecular weight separation range from 10 kDa to 600 kDa (e.g., Superdex 200 16/60 or 26/60) equilibrated with SEC buffer. Monodisperse fractions are pooled, concentrated, and flash-frozen with liquid nitrogen.
3.7 Determination of protein concentration For measuring protein concentration, several popular methods can be applied like absorbance measurement at 280 nm, Bradford assay (Bradford, 1976), or BCA assay (Smith et al., 1985). The latter is used to determine protein concentration of the QFR as detergent interferes with both the absorbance measurement at 280 nm and the Bradford assay. The BCA assay is performed as described in the manufacturer’s manual (Pierce™ BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL, USA).
3.8 Functional characterization Functional characterization of proteins is performed in many ways. In this chapter, the functional characterization of the QFR of W. succinogenes is demonstrated on measuring fumarate reductase or quinol oxidation activity (Unden & Kr€ oger, 1981). This can be performed either in detergentsolubilized state (Lancaster et al., 2000) or in liposomes (Biel et al., 2002; Madej et al., 2006). The latter method was used to provide experimental evidence for the “E-pathway” (Lancaster, 2002b) for essential transmembrane proton transfer in the QFR of W. succinogenes by incorporating a “E-pathway”-defective variant into proteoliposomes (Madej et al., 2006).
3.9 Reconstitution of enzymes in proteoliposomes Reconstitution of purified enzymes of the anaerobic respiratory chain in proteoliposomes is performed according to Biel et al. (2002) and Madej et al. (2006) and comprises two steps: the preparation of sonicated liposomes and the proteoliposome reconstitution procedure. This procedure ensures a unidirectional incorporation of the enzyme into the liposomal membrane where the hydrophilic A and B subunits point outward.
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3.9.1 Material – Bio-Beads SM-2 (Bio-Rad) – Chloroform/methanol solution (2:1, v/v): Mix 6 mL chloroform with 3 mL methanol – n-Dodecyl-β-D-maltopyranoside (Anatrace) – HEPES–KCl: HEPES (5 mM) pH 7.5, KCl (100 mM) – Menaquinol (0.1 M) in ethanol (100%) – 1,2-Dipalmitoyl-sn-glycero-3 phosphocholine (phosphatidylcholine) and 2-dipalmitoyl-sn-glycero-3-phosphate (phosphatidate) (Avanti Polar Lipids, Inc.) 3.9.2 Preparation of liposomes For 5 mL of a 10 liposome stock solution: 1. Weight in 50 mg phosphatidylcholine and 5 mg phosphatidate directly into a 100-mL septum flask. 2. Dissolve lipids in 9 mL of a chloroform/methanol mixture (2:1, v/v) under stirring at room temperature. 3. Add 18.5 μL of a menaquinol stock solution (100 mM) to incorporate a quinol into the liposomal membrane. 4. Tightly close the septum flask and evaporate solvents completely by evacuating. 5. Resuspend the dry phospholipid film (layer) in 5 mL of HEPES/KCl buffer and pour the liposome suspension (11 g phospholipids/L) into a 15-mL Falcon. 6. Sonicate the liposome suspension on ice until it is completely clear (Bandelin Sonopuls equipped with microtip, 4 °C, 30 W, 40% cycle). 7. Make 1:10 dilutions with HEPES/KCl buffer in 5 mL volume in a new Falcon (1.1 g phospholipids/L). 8. Store liposomes at 20 °C until further use. 3.9.3 Preparation of proteoliposomes 9. Thaw 1 liposome suspension and sonificate on ice for 10–15 min (Bandelin Sonopuls equipped with microtip, 4 °C, 30 W, 40% cycle). 10. Transfer liposome suspension into a new 10-mL septum flask and add 0.8 g β-dodecyl-maltoside/g phospholipid and stir carefully for 3 h at room temperature. 11. Add enzyme (0.18 mg/mg phospholipids) stepwise under constant stirring for 1 h at room temperature.
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12. Remove detergent by addition of Bio-Beads SM-2 (240 mg/mL) under stirring for 1 h. 13. Transfer liposome solution into a new Falcon and remove Bio-Beads by centrifugation (5000 g, 30 s) and/or filter-sterilize (0.2 μm pore size). 14. Concentrate proteoliposomes by centrifugation at 17,000 g for 1 min. 15. Discard supernatant and resuspend pellet in 50–100 μL HEPES/ KCl buffer.
3.10 Enzymic assays 3.10.1 Material – UV–VIS Diode Array Spectrophotometer equipped with a multicell transport connected to a water bath (e.g., 8453 UV–VIS Diode Array System Agilent Technologies Inc., Santa Clara, CA, USA) – 25- or 50-μL and 1-mL Microliter syringes with needle (e.g., Hamilton Bonaduz AG, Bonaduz, Swiss) – 1-mL Quartz cuvettes (e.g., macro cells, Agilent Technologies Inc., Santa Clara, CA, USA) – Airtight plugs (Rotilabo plugs, Roth, Karlsruhe, Germany) – Potassium phosphate buffer (50 mM), pH 7.4: Filter-sterilize and degas in a septum flask – Tris buffer (50 mM), pH 8.0: Filter-sterilize and degas in a septum flask – 2,3-Dimethyl-1,4-naphthoquinone (DMN) (20 mM): Synthesized as described (Lancaster et al., 2005) – Dissolve in ethanol, protect from light, and keep on ice. Do not degas – Borohydride (20 mg/mL): Degas flask with potassium borohydride before dissolving in anaerobic ddH2O. Do not degas afterward – Benzyl viologen (0.1 M): Dissolve in anaerobic ddH2O – Sodium dithionite (50 mg/mL): Dissolve in ddH2O – Fumarate (1 M): Dissolve in H2Odest in a septum flask and degas – Fumarate (0.1 M): Dissolve in H2Odest in a septum flask and degas – Succinate (1 M): Dissolve in ddH2O in a septum flask and degas 3.10.2 Methods All enzymatic assays (Unden et al., 1980) were performed at 37 °C in 50 mM phosphate buffer, pH 7.4 in anaerobized cuvettes (path length 0.4 cm) in the presence of 150 μM DMN (Lancaster et al., 2005). Then 4–8 μL of
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the enzyme (target concentration 5–12 μg/mL) is added and the samples are incubated for 90 s prior to the measurement. 3.10.3 Measurement of the fumarate reduction activity (benzyl viologen ! fumarate assay) The fumarate reduction activity is measured using the artificial substrate benzyl viologen. It is an assay measuring the fumarate reduction activity of the two soluble subunits A and B independent of the membraneembedded C subunit. Benzyl viologen, a colorless substrate in the oxidized form, is reduced by additions of small aliquots of dithionite forming a dark violet color. As it binds artificially to the B subunit of the QFR, it serves as an electron donor for reducing fumarate. The reaction is started with the addition of fumarate and the activity is monitored by measuring the change of the absorbance at λ ¼ 546 nm (ε546 ¼ 19.5 mM1 cm1, d ¼ 0.4 cm). 1. Fill 980 μL of phosphate buffer (50 mM) pH 7.4 (or 50 mM Tris pH 8.0 in case of the SdhABE complex) in an anaerobic cuvette with a microliter syringe. 2. Add 10 μL of a benzyl viologen solution (0.1 M). 3. Reduce the compound by adding 2–5 μL of a sodium dithionite solution (40 mg/mL); absorbance at λ ¼ 546 nm should reach a value of 1.4. 4. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized. 5. Start the reaction with 10 μL of fumarate solution (1 M). 6. Calculate the initial slope of the spectrum after fumarate addition over 10 s. 3.10.4 Measuring the quinol oxidation activity (DMNH2 ! fumarate assay) The assay of quinol oxidation by fumarate is performed according to Unden and Kr€ oger (1981). The measurement is based on the quinol oxidation activity by simultaneous reduction of fumarate. In contrast to the fumarate reduction, the quinol oxidation is strictly dependent on the complete enzyme including the C subunit. As a quinol is needed, the synthetic menaquinone analogon DMN has to be pre-reduced by the addition of small aliquots of a NaBH4 solution. After starting the reaction by addition of fumarate, the reoxidation of DMNH2 is monitored spectrometrically by recording the absorbance difference at λ ¼ 270 and 290 nm (ε270–290 ¼ 15.2 mM1 cm1, d ¼ 0.4 cm). 1. Fill 975 μL of phosphate buffer (50 mM) pH 7.4 in an anaerobic cuvette with a microliter syringe.
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2. Add 10 μL of a DMN solution (20 mM). The absorbance at λ ¼ 270 nm should reach a value of 1.2. The absorbance at λ ¼ 290 nm should remain constant during the complete recording. 3. Pre-reduce the quinone by adding 2.5–5 μL of a potassium borohydride solution (20 mg/mL). The absorbance at λ ¼ 270 decreases to a value of 0.3. 4. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized. 5. Start the reaction with 10 μL of fumarate solution (0.1 M). 6. Calculate the initial slope of the difference spectra after fumarate addition over 10 s. 3.10.5 Measuring the quinone reduction activity The QFR as a SQOR is capable of reducing quinones by succinate oxidation. To measure the quinone reduction activity, the synthetic menaquinone analogon DMN is reduced by addition of succinate. The procedure is basically the same as in 3.10.4 except that the quinone is not pre-reduced by addition of potassium borohydride and succinate is used instead of fumarate. 1. Fill 970 μL of phosphate buffer (50 mM) pH 7.4 in an anaerobic cuvette with a microliter syringe. 2. Add 10 μL of a DMN solution (20 mM). The absorbance at λ ¼ 270 nm should reach a value of 1.2. The absorbance at λ ¼ 290 nm should remain constant during the complete recording. 3. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized. 4. Start the reaction with 10 μL of succinate solution (1 M). 5. Calculate the initial slope of the difference spectra after succinate addition over 10 s. 3.10.6 Calculating of the relative catalytic activity The relative catalytic activity is a useful measure for comparing protein activities. It is the ratio of the specific quinol oxidation activity and the specific fumarate reduction activity. The quotient obtained for the wild-type or reference protein is set to 100% allowing the comparison of RCA values of variants independent of preparation quality.
ACKNOWLEDGMENTS We thank all our collaborators as specified in our cited publications, in particular Hanno Juhnke, Mauro Mileni, and J€ org Simon for their contributions. Support of our research by the Deutsche Forschungsgemeinschaft (DFG, grants INST 256/275-1 FUGG and
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256/299-1 FUGG, SFB 472-Lancaster, GK 845-Lancaster, and GK 1326-Lancaster), the state of Saarland (grant LFFP 11/02), and Saarland University is gratefully acknowledged.
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