2.5.1 Protein Over-Expression
Proteins were expressed (from gene constructs in Table 2.4.8.1 above) in E. coli strains grown in LB media, at levels amenable to purification by Immobilised Metal Affinity Chromatography (IMAC, see section 2.6.6). Increased levels of soluble protein was achieved by modifying these protocols, and allowed expression of both proteins in M9 minimal media. Recombinant proteins were over-expressed in a number of E. coli strains (Table 2.2.2.1) using IPTG induction. Strains were made competent (section 2.2.3) and transformed with an expression plasmid containing the recombinant gene (section 2.3) (in some cases with a second plasmid to aid expression) and plated on LB agar plates with appropriate antibiotics.
(i) Overexpression in LB media
A single colony was picked for a small-scale pre-culture and grown, at 37oC, 180 rpm, overnight in 5 or 10 ml of LB media and appropriate antibiotics. The pre-culture was diluted at 1 in 50 or 1 in 100 into LB media containing the appropriate antibiotics (and 0.2% glucose for VanSA), and grown at 37
o
C, 180 rpm, before induction with IPTG to a final concentration of 0.2 mM or 1 mM when the optical density of a culture, at a wavelength of 600nm, was between 0.5 and 0.6. Expressions were initially grown for 4 hours at 37oC, but it was found that 12-16 hours at 25oC (for VanSA) or 20-24 hours at 16
o
C (for VanSSC) gave higher yields
of soluble protein, before harvesting at 10,000g, 15 mins using a Beckmann JA14 rotor.
Cell pellets were washed in ice-cold phosphate-buffered saline (PBS) and the pellet was resuspended in 3 mL of resuspension buffer per gram of wet cell weight. To prevent protein degradation, protease inhibitors (2μM leupeptin, 2μM pepstatin, 0.2mM PMSF final concentrations) were added. Resuspended cells were snap-frozen and stored at -80oC.
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(ii) Overexpression in M9 Minimal Media
Both pProEx::vanSA and pProEx::vanSSC were transformed onto LB plates containing 100
μg/ml of ampicillin and 35 μg/ml of chloramphenicol (see section 2.3). A scraping of colonies was taken using an inoculation loop, and added to 10 mL starter cultures in LB containing the appropriate antibiotics with addition of 2 g/L glucose for those containing
vanSA. Starter cultures were grown at 37
o
C for 12-14 hours and used to inoculate 800mL cultures at a starting optical density at 600nm of ~0.01. These larger cultures were grown at 37oC until an optical density at 600nm of ~ 0.7-0.8, at which point media was switched to M9 (see section 2.2.1) from LB, by pelleting at 3000 g for 10 minutes in a Beckmann benchtop centrifuge, washing and resuspending twice in M9 media. In the final resuspension step, the pellet was resuspended in either 800ml (‘1x’), 400ml (‘2x’), or 200ml (‘4x’) of M9 minimal media containing: 1x BME vitamins (Sigma), 1 g/L unlabelled or 15N-labelled NH4Cl, 4 g/L glucose, 0.1 mM CaCl2, 2mM MgSO4, 50 μM FeCl3, 1x M9 salts, 100 μg/ml
Ampicillin, 35 μg/ml Chloramphenicol and pH adjusted to 7.5 or 8.0 for higher buffering capacity.
Cells were incubated at the induction temperature for 1-2 hours prior to induction to discharge unlabelled metabolites, and overexpressed with IPTG for 16-20 hours (0.2 mM IPTG and 25oC for VanSA and 1mM IPTG and 18oC for VanSSC). Cultures were then
pelleted at 3000 g for 10 minutes, and aliquots taken for SDS-PAGE (section 2.6.1) and Western Blot analysis (section 2.6.3). Cell pellets were resuspended in 10 mL/L of
resuspension buffer, and to prevent protein degradation, protease inhibitors (2μM leupeptin, 2μM pepstatin, 0.2mM PMSF final concentrations) were added. Resuspended cells were snap-frozen and stored at -80oC.
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2.5.2 Preparation of Membrane Protein Lysates
For small-scale production, membrane protein lysates were prepared, as described in Ward et al. (2000), using the ‘Water Lysis’ method. 100 mL of an overnight expression (section 2.5.1) was centrifuged at 10,000 g, 10 mins at 10oC in a JA14 rotor with the cell pellet resuspended in 0.2 M Tris-HCl (pH 7.8). At time zero, 9.7 mL of sucrose buffer (1M sucrose, 1mM EDTA, 0.2M Tris-HCl, pH7.8) was added, followed by 1 mL of 1.3 mg/mL lysozyme at 1.5 minutes and 20 mL of deionised water at 2 minutes and left to shake for one hour at 25oC to form spheroplasts, due to the high osmotic shock experienced by the cells. Spheroplasts were sedimented at 20,000 g, 20 mins at 10oC in a Beckman JA25.50 rotor. The pellet was re-suspended, using a hand-held homogeniser, in 30 mL of water until cloudy and no lumps present and left to stand for 30 minutes at 25oC.
Membranes were sedimented at 30,000 g, 20 mins at 4oC in a JA 25.50 rotor and the
resulting pellet resuspended in membrane resuspension buffer (0.1 M Sodium phosphate, pH 7.2, 1 mM 2-mercaptoethanol). Membranes were washed twice in 15 mL of membrane resuspension buffer using a hand-held homogeniser each time to resuspend the pellet. Finally washed membranes were resuspended in 1 – 5 mL (depending on the weight of protein obtained) of membrane resuspension buffer containing 1 mM MgCl2 to which DNase was
added to a final concentration of 20 μg/mL. Preparations were incubated at 37oC for 30 minutes before addition of EDTA to a final concentration of 1 mM. Preparations were analysed using SDS-PAGE (section 2.6.1) and stored at -80oC.
2.5.3 Large-Scale Membrane Protein Preparations
Large-scale membrane protein preparations followed the protocol given by Quigley (2010). The expressed cell pellet (from one or more litres of culture) was mixed with resuspension buffer (20 mM HEPES pH 7.8 or 50 mM phosphate buffer pH 7.5, 300 mM NaCl) in the
[74]
ratio of 3mL per gram of wet cell pellet, before snap-freezing and storing at -80oC. Upon thawing, DNase (20 μg/ml final concentration, Roche), magnesium chloride (10 mM final concentration) and chicken egg lysozyme (2.5 mg/ml final concentration, Calbiochem) were added. After rocking at 4oC for 30 mins, cells were lysed by passing through a continuous cell disrupter (Constant Cell Disruption Systems) three times at 30 kpsi and 4oC. Lysate was centrifuged at 10,000g for thirty minutes using a Beckman JA14 rotor to pellet cell debris before the supernatant was transferred to ultracentrifuge tubes and centrifuged at 40 krpmfor two hours using a Beckman Ti45 rotor in an ultracentrifuge (Beckman Optima L90K) to pellet the membrane fraction. Membranes were resuspended in 20 mM HEPES pH 7.8, 300 mM NaCl, 20 mM Imidazole and 20% Glycerol, in 10 mL of buffer per litre for VanSA or
per three litres for VanSSC, before storing at -80 o
C.
2.5.4 Protein Solubilisation using Membrane Mimetics
Membrane proteins are naturally embedded in a complex and dynamic lipid bilayer, limiting their analysis by standard biophysical techniques. In vitro studies are reliant on successful solubilisation of membrane proteins, using appropriate membrane mimicking environments. These must consist of a solubilising component and satisfy the hydrophobic nature of TM segments, while bringing loop regions into contact with an aqueous phase. Therefore lipid and detergent systems are often chosen.
Detergents are amphipathic with polar head groups and a single hydrophobic tail, and can spontaneously form spherical, micellar structures (Seddon et al., 2004) above a Critical Micelle Concentration (CMC) (Bhairi, 1997). The number of solubilised monomers contained in a micelle is known as the mean aggregation number (a), and is related to bulk molar detergent concentration (d) and micelle concentration (m) by Equation 1.
[75] d C m a (1)
Detergents tested include dodecylphosphocholine (DPC) (a = 56 and CMC = 1.1-1.2 mM) (Wüthrich, 1986), dodecylmaltoside (DDM) (a = 98 and CMC = 0.17-0.3 mM),
octylglucoside (OG) (a = 84, CMC = 18-20 mM) (Bhairi, 1997) and sodium dodecyl sulphate (SDS) (a = 62 and CMC = 8-14 mM, at 298K) (Nishikido, 1983). A full list of all detergents tested is given in Appendix Table 1.
Prepared membranes were thawed and detergent added to a final concentration of 0.5-1% w/v, before membranes were incubated at 4oC on a roller rocker for two hours. Solubilised membranes were centrifuged at 40 krpm for 45 mins using a Beckman TLA 100.3 rotor and benchtop ultracentrifuge (< 20ml volume) or Beckman Ti45 rotor and Beckmann T90K ultracentrifuge (< 350ml volume). The resulting supernatant was added to charged nickel, high performance agarose (Qiagen) or sepharose (GE Healthcare) slurry (ratio of 1mL slurry per mg of expected VanSprotein) pre-equilibrated in 20 mM HEPES pH 7.8, 300 mM NaCl, 10% Glycerol, 20 mM Imidazole, 0.1% w/v detergent). This protein-resin mix was left to rock for 1-2 hours at 4oC before pouring into a column and allowing the resin bed to settle.