Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions
Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies.
KeywordsHistidine kinase Membrane proteins Vancomycin resistance Enterococci Pheromone sensors Streptococcus pneumoniae Circular dichroism spectroscopy Analytical ultracentrifugation
Histidine protein kinase
Extracellular sensing domain
Membrane proteins are crucially important for the physiological functioning of biological cells, possessing roles in molecular recognition and signal transduction, transport of essential nutrients, exclusion of toxic molecules, ion regulation and energy generation. Between 20 and 30% of proteins encoded by eubacterial, archaeon and eukaryotic genomes are membrane proteins (Wallin and von Heijne 1998), and in humans defects in their proper folding or other mutations can result in many serious diseases. Approximately, 60% of current small molecule drugs target membrane proteins (Terstappen and Reggiani 2001; Davey 2004; Arinaminpathy et al. 2009), testifying to the potential of identifying further membrane proteins as new drug targets in the future. Knowledge of the three-dimensional structure of membrane proteins coupled with how they function is one important area for drug discovery goals. But largely due to their hydrophobicity, membrane proteins pose technical challenges for their expression and purification and for structural work such as crystallisation. As a result, only approximately 2% of protein structures in the Protein Data Bank (PDB) are membrane proteins (http://blanco.biomol.uci.edu/mpstruc/; Miles and Wallace 2016).
Yet, there have been recent advances in methods for the production of low abundance membrane proteins, including bacterial membrane proteins (e.g. Potter et al. 2002; and reviewed in Saidijam et al. 2003, 2005; Suzuki and Henderson 2006; Moraes et al. 2014; Hardy et al. 2016; Hussain et al. 2016; Lee et al. 2016; Lee and Pollock 2016; Rawlings 2016), leading to increased successes in crystallisation, structural determination and in elucidation of mechanisms of action (e.g. Weyand et al. 2008; Shimamura et al. 2010; Simmons et al. 2014). Many of the expression and purification strategies for bacterial membrane proteins are based on work with highly hydrophobic membrane transport proteins (Ward et al. 1999; Saidijam et al. 2005; Suzuki and Henderson 2006). Here, we present an adapted strategy based on these established methods for reliable production of membrane histidine protein kinases (HPKs) of bacterial two-component signal transduction systems (Potter et al. 2002; Ma et al. 2008) and a variety of other membrane proteins involved in antimicrobial resistances (AMR) and peptide pheromone sensing, described below.
Resistance by bacteria to current antibiotic agents is recognised to be a major global problem in the treatment of hospital and other infections. Resistance mechanisms (which can be considered as nanomachines) mounted by bacteria in response to exposure to antibiotic drugs often comprise cascades of molecular mechanisms for drug recognition, signal transduction and production of efflux and/or deactivation processes to remove the antibiotic threat and ensure bacterial survival (Phillips-Jones & Harding 2018). Many of the components involved in antibacterial resistances are membrane proteins. Structural elucidation of these components and characterisation of their mechanisms of action are important for future intervention strategies. Biophysical approaches also provide important information. For example, we recently used hydrodynamic methods to demonstrate binding by the glycopeptide antibiotic vancomycin to the VanA-type VanS histidine kinase membrane receptor (Phillips-Jones et al. 2017a, b). Furthermore, using circular dichroism spectroscopy, we showed that glycopeptide binding to VanS is weak (Hughes et al. 2017). Intact VanS protein was produced for these experiments through the commonly used method of heterologous expression of a plasmid-borne gene (in this case vanS) in a heterologous Escherichia coli host, using a membrane protein overexpression plasmid such as pTTQ18His (e.g. Saidijam et al. 2005). However, modifications to these published strategies for expression and purification were necessary to ensure success. Furthermore, following VanS purification, reduced or no detergent was added for subsequent biophysical experiments to prevent potential inhibition of dimerization and phosphorylation activities, as commonly observed for many HPK proteins. Here, we evaluate these modifications that proved successful for production of active intact VanS, for the expression of a collection of HPK and AMR membrane proteins of different membrane topologies, and for two specific examples of peptide pheromone-sensing HPKs (BlpH and ComD of Streptococcus pneumoniae (de Saizieu et al. 2000; Pestova et al. 1996; Cheng et al. 1997; Podbielski & Kreikemeyer 2004), including their ligand binding abilities post-purification. We compare this adapted strategy with that used previously for other Enterococcus faecalis HPKs by Ma et al. (2008), who reported the successful expression in E. coli of 15 out of 16 of the genome complement of membrane HPK genes and the purification of 12 out of the 15 expressed HPKs. We also investigate another strategy for investigations of ligand binding and sensing mechanisms using only the predicted sensory domain in isolation; we sought to determine whether the soluble extracellular sensory domain of the Enterococcus faecalis VicK histidine kinase, VicKESD, lacking its transmembrane segments can be successfully expressed and purified for future characterisation using biophysical techniques.
Materials and methods
Genes involved in conferring resistance to glycopeptide antibiotics in enterococci
Plasmids were constructed, sequenced and kindly provided by Dr Djalal Meziane-Cherif and Professor Patrice Courvalin (Pasteur Institute, France). The Enterococcus faecalis BM4518 vanTG (encoding VanG-type serine racemase involved in transport and conversion of l-serine to d-serine) (AAQ16274.1) and vanTG-M (encoding residues 1–342 of VanTG—the membrane serine transporter component of VanTG) (Meziane-Cherif et al. 2015) were cloned into the membrane protein overexpression plasmid pTTQ18His as described in Ma et al. (2008). Cloning and expression of the gene encoding the VanA-type VanS membrane histidine kinase of E. faecium B4147 has been described previously (Phillips-Jones et al. 2017a). The VanA-type vanZ gene of E. faecium B4147 (CDD 121006; AAA65959 Arthur et al. 1995), which confers teicoplanin resistance, was cloned into pMR2, a derivative of pTTQ18His, introducing a His6 tag at the N-terminus of the expressed recombinant protein ensuring a predicted location for the His6 tag on the inside of the membrane (Rahman et al. 2007).
Streptococcus pneumoniae ComD and BlpH pheromone-sensing histidine protein kinases
The blpH (de Saizieu et al. 2000) and comD (Cheng et al. 1997; Pestova et al. 1996) genes of S. pneumoniae ATCC700669 were amplified by PCR and cloned into pTTQ18His as described previously (Ma et al. 2008). The comD gene was cloned as a SacI-SalI ended fragment, whilst the blpH gene was cloned as an EcoRI-PstI-ended fragment. Clones were verified by gene sequencing. An RGS(H)6 sequence was introduced at the C terminus of both proteins to facilitate purification by nickel affinity methods (Ma et al. 2008).
Extracellular sensing domain of enterococcal VicK
The putative extracellular sensing domain of VicK, VicKESD (equivalent to residues 35–176 of E. faecalis V583 vicK), was amplified by PCR and cloned as an NdeI-BamHI fragment into pET14b. The correct sequence was verified by DNA sequencing. The expressed protein with an N-terminal GSS(H)6SSGLVPRGSHMI sequence was predicted to be 18,238 Da and this was confirmed by mass spectrometry.
All expression plasmids were transformed into E. coli BL21 [DE3].
For small-scale expression trials of vancomycin resistance genes, cultures (50 ml) were grown aerobically in Luria–Bertani (LB) broth containing 100 μg ml−1 carbenicillin at 37 °C until an absorbance (A600) of 0.5 was reached when 1 mM isopropyl β-d-1-thiogalactoside (IPTG) was added for induction of van gene expression (Ward et al. 1999; Saidijam et al. 2005). Growth was permitted to continue for a further 3 h post-induction at a reduced temperature of 30 °C before cell harvesting. No optimisations of expression conditions were undertaken. Mixed E. coli membranes were prepared by the water lysis method (Ward et al. 1999) and samples analysed by SDS-polyacrylamide gel electrophoresis and Western blotting (see below).
Expression of S. pneumoniae comD and blpH was undertaken in large-scale (6 l) culture experiments employing the non-optimised strategy described above. Briefly, E. coli BL21 [DE3] cells harbouring pTTQcomD or pTTQblpH were cultured aerobically in 6 l of selective Luria–Bertani (LB) broth at 37 °C and induced with 1 mM IPTG. The cultures were incubated for a further 3 h at a reduced temperature of 30 °C prior to cell harvesting. Cells were lysed by explosive decompression and mixed membranes prepared as described by Ma et al. (2008).
Expression trials of soluble VicKESD was undertaken by cultivation of E. coli BL21 [DE3]/pET14b-VicKESD in 500 ml of LB broth containing 100 μg.ml−1 ampicillin at 37 °C and induction with 0.4 mM IPTG induction as described above for the Van proteins. After incubation for a further 3 h at 37 °C, cells were lysed by sonication and the insoluble membrane fraction harvested by centrifugation at 100,000g for 40 min at 4 °C. Separated soluble and insoluble fractions were stored at − 20 °C prior to SDS-PAGE analysis.
All growth experiments included control cultures in the absence of IPTG (uninduced cultures).
Purification of BlpH, ComD and VicKESD proteins
For BlpH and ComD membrane proteins, total mixed membranes of E. coli BL21 [DE3] harbouring pTTQblpH or pTTQcomD were prepared using methods described previously (Potter et al. 2002; Ma et al. 2008), except that the sucrose density gradient centrifugation step used to separate the inner and outer membranes was omitted. His-tagged BlpH and ComD proteins were solubilised from mixed membranes using 1% (w/v) DDM detergent and purified by nickel affinity chromatography as described by Ma et al. (2008) using wash buffers containing 20 mM imidazole and elution buffers containing 200 mM imidazole, both of which contained 0.05% n-dodecyl-β-d-maltoside (DDM) (Saidijam et al. 2005). Purified proteins were exchanged into 10 mM sodium phosphate, pH 7.2, containing 0.05% DDM.
For purification of VicKESD protein from soluble cell fractions, nickel affinity chromatography was undertaken in the absence of DDM, but otherwise according to Ma et al. (2008) using wash buffers containing 20 mM imidazole and elution buffer comprising 200 mM sodium acetate, pH 4.0. Purified protein was exchanged into 20 mM Tris–HCl, pH 7.9, or 20 mM sodium acetate, pH 4.0, or 20 mM sodium phosphate, pH 7.2, or 10 mM ammonium hydrogen carbonate, pH 8.0, using Centricon filter devices.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Proteins were separated by SDS-PAGE using 4–5% stacking and 12% resolving SDS-polyacrylamide gels prepared and subjected to electrophoresis according to Sambrook et al. (1989). For Western blotting experiments, the separated proteins were first electroblotted onto Fluorotrans Transfer membrane (Pall Corp.) as described previously (Ma et al. 2008).
Western blotting experiments to detect the presence of the His6 tags of recombinant overexpressed proteins were performed using the INDIA™ HisProbe-HRP (Perbio Science UK Ltd) with Supersignal West Pico Chemiluminescent substrate (Pierce) detection as described previously (Ma et al. 2008).
To confirm the molecular masses of purified VicKESD and VanS, the electrospray ionisation mass spectrometry facility of the Astbury Centre for Structural Molecular Biology, University of Leeds, UK, was used (Phillips-Jones et al. 2017a).
Purified proteins (VanS and VicKESD) were transferred to Fluorotrans Transfer membrane (Pall Corp.), visualised by Coomassie Blue G-250 staining and bands excised for sequencing using Edman degradation (Alta Biosciences, University of Birmingham, UK).
Protein determinations were carried out using bicinchoninic acid in a Pierce™ BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. Bovine serum albumin was used as standard.
BlpH and ComD ligands
Circular dichroism spectroscopy (CD)
CD experiments were performed using a nitrogen-flushed Jasco J715 spectropolarimeter (for VicKESD) or an Applied Photophysics Chirascan Plus instrument (for ComD and BlpH). Purified membrane proteins were typically prepared in 10 mM sodium phosphate, pH 7.2–7.6 (in the presence of 0.05% DDM for membrane proteins) (Patching et al. 2012; Miles and Wallace 2016) and allowed to equilibrate for 20 min at 15 or 20 °C prior to acquisition of spectral data. A bandwidth of 2 nm was used and data acquired at 1 s/nm. No data in which the HT of the detector exceeded 600 V were included in the analyses.
For CD measurements in the far-UV (180–260 nm), protein concentrations in the range 2.4–14.0 μM (~ 0.12–0.25 mg ml−1) were employed in sample volumes of 200–350 μl using a pathlength of 1 mm. The longer pathlength cell was selected on the basis of the buffer and detergent concentrations used here (Miles and Wallace 2016). For VicKESD measurements at 15 and 37 °C, 30 scans were obtained; for thermal denaturation experiments, 5 scans were obtained. For BlpH and ComD proteins, spectra are the average of two scans. Thermal denaturation experiments for VicKESD were performed at a starting temperature of 15 °C which was increased stepwise and incrementally to 90 °C; at each step, 10 min equilibration time was permitted before acquisition of spectral data.
For CD measurements in the near-UV region (250–340 nm), protein concentrations of 3.6 µM (0.2 mg ml−1) (ComD) or 9.5 µM (BlpH) were employed in sample volumes of 400 µl using a pathlength of 10 mm. Spectra shown are the average of ten scans. Reaction mixes were incubated with peptide ligands (or equivalent volumes of acetonitrile solvent) for 20 min prior to acquisition of spectral data as described by Patching et al. (2012).
In all cases, control spectra of buffers with other relevant additives (in the absence of added proteins) were also obtained. These spectra were subtracted from spectra obtained for each purified protein and used to derive difference spectra. The maximum concentration of acetonitrile in ligand experiments was 0.45%.
Expression of VanS, VanTG, VanTG-M and VanZ membrane proteins involved in vancomycin resistance
Additional bands in induced cultures harbouring pTTQ-vanTG-M and pTTQ-vanS were also visible in the SDS-PAGE gels (Fig. 1a) indicative of higher levels of overexpression. No signals were detected in any of the uninduced control membranes, indicating tight regulatory control of the ptac promoter of pTTQ18His in the absence of inducer, which is consistent with previous observations (Ma et al. 2008). The predicted masses of the His-tagged proteins are 81.2 kDa (VanTG), 38.8 kDa (VanTG-M), 45.8 kDa (VanS) and 20.1 kDa (VanZ). The positive protein bands shown in Fig. 1 appear of lower mass than these predicted values. This is likely to be due to anomalous migration behaviour that is typical of hydrophobic membrane proteins in the approximate technique of SDS-PAGE. However, final confirmation that the detected protein bands are indeed intact VanTG, VanTG-M and VanZ would routinely be carried out following purification of each protein via mass spectrometry and/or N-terminal sequencing (see Ma et al. 2008 for example). Indeed, the intact nature of the VanS protein has already been confirmed in our laboratory; VanS was purified as described in Phillips-Jones et al. (2017a) and a mass of 45,775 Da was determined using electrospray ionisation mass spectrometry (ESI-MS) which closely matches the predicted mass of 45,765 Da. Further confirmation was provided by sedimentation equilibrium experiments, which determined the overall weight average molar mass to be 46.4–47.7 kDa (Phillips-Jones et al. 2017a). N-terminal sequencing confirmed the presence of the expected n-MNSHM sequence and the protein was shown to retain its autophosphorylation activity post-purification (Phillips-Jones et al. 2017a).
Expression, purification and verification of S. pneumoniae BlpH and ComD histidine protein kinases
Ligand binding studies using purified BlpH and ComD
Cloning, overexpression and purification of VicKESD, the extracellular sensory domain of VicK of E. faecalis V583
Structural status and thermal denaturation studies of VicKESD investigated using far-UV CD spectroscopy
To the best of our knowledge, this is the first study to successfully express and purify the intact versions of the S. pneumoniae BlpH and ComD (pherotype 2) membrane sensor kinases. A previous study utilised domain fragments lacking the transmembrane domains (Sanchez et al. 2015). The successful expression and purification of these two membrane HPKs (Figs. 2, 3) adds to the high success rate of expressing members of this membrane protein family originating from Gram-positive species in the membranes of a Gram-negative E. coli host. A total of 15 out of the 16 membrane HPKs from E. faecalis were successfully expressed previously, many of which were expressed using a strategy similar to that proposed here (Ma et al. 2008). In addition, 12 of the 15 expressed proteins were successfully purified (Ma et al. 2008). All purified HPKs tested so far, including ComD and BlpH in the present study, possessed structural integrity post-purification as revealed by far-UV CD spectroscopy (Fig. 4). In addition, purified ComD and BlpH interacted with and bound their peptide pheromone ligands CSP2 and BlpC, as revealed in the near-UV CD difference spectral measurements (Fig. 4). Together with previous successes in ligand binding studies using the same approach with other intact sensor kinases [e.g. FsrC (Patching et al. 2012) and VanS (Phillips-Jones et al. 2017a)], these results further strengthen the promise and potential of the in vitro approach described here for routine identification of ligands and ligand screening methods. Of particular interest is the finding here that the CSP1 pheromone, as well as the native CSP2 ligand, interacted with ComD2 (Fig. 4d). CSP1 and CSP2 belong to two distinct and major pherotypes of S. pneumoniae (pherotypes 1 and 2, respectively) and are proposed to exhibit specific interactions only with their cognate ComD HPK of their respective pherotypes, thereby inducing competence for their own specific pherotype only (Pozzi et al. 1996; Iannelli et al. 2005; Johnsborg et al. 2006; Allan et al. 2007; Shpakov 2009). Consistent with that evidence, it is possible that the binding to ComD shown in the present study by both CSPs (which exhibit approximately 50% sequence identity) also occurs in vivo resulting in different conformational change outcomes (consistent with Fig. 4d data), with only that due to CSP2 binding resulting in downstream activation of ComD and ComE and thence induction of competence. However, there are other possibilities. For example, some S. pneumoniae strains/pherotypes have been reported to be inducible for competence by both CSP1 and CSP2 (Pozzi et al. 1996), which would suggest that strain ATCC 700669 used here might provide another example of such strains. Alternatively, it has been shown that high concentrations of non-cognate CSP1 can induce competence in pherotype 2 strains in vitro (Iannelli et al. 2005). These possibilities are now being investigated.
Batch growth of at least 6 l of E. coli BL21 [DE3] host bacterium harbouring pTTQ18His or pMR2 expression plasmids with the cloned HPK gene. In our experience, the most suitable culturing conditions are aerobic culturing in Luria–Bertani (LB) selective broth [without recourse to minimal or very rich media used previously for expression of members of some other membrane protein families, but which in our hands usually produces lower expression levels of HPKs (unpublished data)].
At mid-exponential phase, addition of 1 mM isopropyl-thiogalactoside inducer of HPK gene expression, followed by a rapid reduction in incubation temperature to 30 °C. Post-induction incubation is usually of 3 h duration, but may benefit from longer time periods for some proteins. Cell harvesting by centrifugation and preparation of mixed E. coli membranes is as described in Ward et al. (1999).
Purification of His6-tagged HPK proteins is by nickel affinity chromatography as described previously (Ward et al. 1999; Ma et al. 2008) using HEPES-based buffers containing 20% glycerol and 0.05% suitable detergent such as n-dodecyl-β-d-maltoside (DDM) in the presence of 20 mM imidazole in wash buffers and 200 mM imidazole in elute buffers. Sodium chloride is usually omitted. During buffer exchanges, the detergent concentration is usually reduced to 0.025% which is still above the critical micelle concentration (CMC) value for DDM (0.17 mM or 0.0087%), yet retains the activity of most HPKs and their ability to dimerize upon ligand binding.
For many years, one of our main aims for expressing and purifying intact membrane HPKs, rather than domain fragments or soluble portions lacking the transmembrane regions, has been to investigate ligand (and inhibitor) binding and the ensuing downstream molecular events that then take place including transduction of the ligand signal (or inhibitor signal) across the membrane to the soluble domains within the cytoplasm involved in phosphorylation (Potter et al. 2002, 2006; Ma et al. 2008, 2011; Patching et al. 2012; Phillips-Jones et al. 2013, 2017a, b). The approach seems logical given that the sensory domains are often located within the transmembrane segments. Yet, such an approach applied to membrane sensor kinases is in some cases much more challenging to undertake than for the more hydrophobic membrane transport proteins for which detergent is absolutely essential to maintain solubility and function. Membrane sensor kinases may possess as few as two transmembrane segments with a much larger soluble portion comprising the kinase and ATP-binding domains that reside in the cytoplasm in vivo. The two predicted transmembrane segments of the A-type VanS make up just 10.8% of the protein. Therefore, for functional studies the large soluble portion as well as the ‘minor’ hydrophobic portion of the transmembrane domain must both be considered. Solubility trials often influence how to proceed—for VanS, solubility was often better when detergent was omitted post-elution (Phillips-Jones et al. 2017a). Use of detergent-less buffers facilitated identification by hydrodynamic methods of vancomycin as a ligand; binding was observed in the absence of detergent. Indeed, VanS by itself had a symmetry of 12:1 in aqueous solvent which then became 5:1 in the presence of ligand, suggesting a significant conformational change and possibly behaviour reminiscent of intrinsic disorder proteins in aqueous solution (Dunker et al. 2002). But the accompanying dimerization event was lacking (Phillips-Jones et al. 2017b), though the protein was shown to be active in activity-based buffers (Phillips-Jones et al. 2017a, b). So, perhaps a detergent or a membrane environment is required after all to observe some events, though in our experience the presence of detergent usually reduces or abolishes HPK autophosphorylation activities. In any case, in the example of VanS in the absence of detergent, ligand identification was successfully achieved.
Another approach for investigations of ligand binding by membrane HPKs is to clone and express the predicted sensing domains in isolation, without the TMs. If the sensing domain is sufficiently large, then this eliminates the challenges posed by the hydrophobic full-length membrane proteins and studies can be pursued in the absence of added detergent. It assumes, however, that the TMs are not involved in the sensing mechanism. This approach was successfully used recently in studies of YycG-ex, the extracellular sensing domain of the S. aureus VicK homologue, YycG. The crystal structure of YycG-ex was determined and proved insightful for determining how the intact protein functions (Kim et al. 2016). Adopting a similar approach here for E. faecalis VicKESD, which possesses a predicted sensing domain of 141 residues, the sensing domain was successfully overexpressed as a recombinant His-tagged soluble protein of 162 residues (lacking the fMet) and approximately 18 kDa (Fig. 5). The purified protein retained structural integrity post-purification and was soluble at concentrations of up to 19 mg/ml suitable for crystallisation and other structural studies. The domain was overall α-helical and exhibited some denaturation upon increases in temperature from 15 to 50 °C, as revealed through measurements of secondary structure composition, but at > 50 °C structural integrity remained relatively stable (Fig. 6).
For successful and routine expression in E. coli of intact bacterial histidine kinase and vancomycin resistance membrane proteins, an adapted strategy of the methods described by Ward et al. (1999) and Saidijam et al. (2005), and described in the present study was shown to be sufficient to obtain the expression of all these membrane proteins. Further optimisations can then be undertaken to maximise membrane protein production, but for the proteins tested here no further optimisations were necessary for obtaining sufficient purified proteins for downstream biophysical characterisations, including identification and confirmation of ligand interactions by ComD and BlpH. Expression of sensing domains in isolation may be another useful approach for producing members of these protein families.
We thank Professor Patrice Courvalin and Dr Djalal Meziane-Cherif (Institute Pasteur, France) for construction and provision of vancomycin resistance expression plasmids, Professor Sheena E. Radford (University of Leeds) for provision of circular dichroism facilities (Wellcome Trust Grant 094232) and Professor Alison E. Ashcroft (University of Leeds) for mass spectrometry facilities. Plasmid pMR2 was kindly provided by the late Professor Stephen Baldwin (University of Leeds). We thank Mr David Sharples (University of Leeds) for use of fermenter facilities for large-scale cultivation of E. coli. We thank Dr Kerry Rostron (University of Central Lancashire) for cloning blpH and comD and for sequence verification. This work was supported by the Biotechnology & Biological Sciences Research Council [grant numbers BB/D001641/1 and BB/M013081/1], the first grant to MKP-J and PJFH, the second grant to MKP-J. MKP-J also thanks the Innovation & Enterprise Office of the University of Central Lancashire for funding. AAA and JMK are grateful to the University of Central Lancashire for internships.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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