2.4.1 Expression of Histidine-tagged Colicin N-TR
Truncated colicin N with a C-terminal His-Tagged (-SSHHHHHH) (ColN-TR) was expressed from E. Coli BL21-AI with the plasmid pET3a. Selected transformed cells from overnight culture were grown in LB (Luria-Bertani) medium containing 100 µg/ml ampicillin at 37 °C. At OD600 = 0.6, L-arabinose
was added to cell culture to a final concentration of 0.2% (v/v) and cells were then grown for a further 3 h. To harvest cells, the cell culture was centrifuged for 10 min at 8,000 g at 4oC.
2.4.2 Expression of Histidine-tagged TolA II-III
Truncated TolA II-III with an N-terminal His-tag was expressed from E. Coli BL21-AI as a host with the plasmid pET8c. Selected transformed cells from overnight culture were grown in LB (Luria-Bertani) medium containing 100 µg/ml ampicillin at 37 °C. At OD600 = 0.6, L-arabinose was added to cell culture to a
final concentration of 0.2% (v/v) and cells were then grown for a further 3 h. To harvest cells, the cell culture was centrifuged for 10 min at 8,000 g at 4oC.
2.4.3 Expression of Histidine-tagged membrane scaffold proteins (MSP1E3D1)
Membrane scaffold protein with an N-terminal His-tags (MSP1E3D1) was expressed using the pET expression system using E. Coli BL21(DE3) as a host. Selected transformed cells from overnight culture were grown in LB (Luria- Bertani) medium containing 30 µg/ml Kanamycin at 37 °C. At OD600 = 0.6, IPTG
(isopropyl β-D-thiogalactoside) was added to the cell culture to a final concentration of 1 mM. After growing for 3 h, cells were harvested by centrifugation for 10 min at 8,000 g at 4oC.
2.4.4 Purification of Histidine-tagged proteins
The cell pellet harvested by centrifugation was resuspended in Ni column loading buffer (50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl, 10 mM imidazole) and then disrupted by sonication on ice for 15 min. To remove unbroken cells and cell debris, the resulting suspension was centrifuged at 17,000 g at 4oC. The clarified supernatant was loaded on to a Ni-NTA (nickel- nitriloacetic acid) column previously equilibrated in Ni column loading buffer. The removal of non-specifically bound proteins was done by washing the column twice with Ni column wash buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 25 mM imidazole). Bound protein was eluted using a 25-250 mM imidazole gradient collecting 5 ml fractions. Protein containing fractions were then dialysed twice against 50 mM sodium phosphate buffer, pH 7.4, 300 mM NaCl.
2.4.5 Expression of wild-type OmpF
WT OmpF was produced from E. coli BE3000 (Garavito and Rosenbusch, 1986). Cells were grown in 2 x 50 ml in LB (Luria-Bertani) medium at 37 °C overnight. The cells from the overnight culture were then harvested by centrifugation at 8,000 g at room temperature and resuspended into 10 ml of fresh LB medium. 10 ml of cell culture was inoculated into a 1.5-litre fermentation vessel in a Minifors fermenter (INFORS) at 37oC using glucose as a carbon source. During cell growth, a pH of 7.3 was maintained by adding 10% (w/v) NaOH. The measurement of OD600 by a UV-1800 UV spectrophotometer
(Shimadzu) was performed to monitor the cell growth. When the OD600 reached
at least 10.0, the cells were harvested by centrifugation at 8,000 g for 10 min at 4oC.
2.4.6 Production of deuterated OmpF
The deuterated OmpF was produced from E. coli BE3000 (Garavito and Rosenbusch, 1986). Cells were first adapted onto a hydrogenated, solid minimal medium plate and followed by growth on a 85% D2O minimal medium
plate (Artero et al., 2005). The recipe for minimal media is shown in Table 2.9. Once colonies grew on the plate (which normally takes a few days due to the slower growth of the cells), selected larger colonies were grown in 50 ml of 85% D2O minimal liquid medium. Once growth is established overnight, these cells
were inoculated at 1:20 ratio into 2 x 50 ml of fresh 85% D2O minimal liquid
medium. This step was repeated three times in order to increase the initial growth rate. Cells were harvested by centrifugation at 8,000 g at room temperature and resuspended into 10 ml of fresh 85% D2O minimal liquid
medium. This cell culture was then inoculated into the 1.5 L fermenter as previously described in section 2.4.5.
Table 2.9 Recipe of minimal media for the production of deuterated OmpF (Meilleur et al., 2009). Component Concentration (NH4)2SO4 3.45 g/L KH2PO4 0.78 g/L Na2HPO4.2H2O 3.24 g/L (NH4)2-H-citrate 0.25 g/L MgSO4.7H2O* 0.13 g/L
Trace metal solution** 0.5 ml/L
Glycerol 2.5 g/L
* MgSO4.7H2O is added last otherwise it will precipitate.
**Trace metal solution contains 0.5 g/L CaCl2.2H2O, 16.7 g/L FeCl3.6H2O, 0.18
g/L ZnSO4.7H2O, 0.16 g/L CuSO4.5H2O, 0.15 g/L MnSO4.4H2O, 0.18 g/L
CoCl2.6H2O, 20.1 g/L EDTA.
2.4.7 Production of mutant OmpF proteins
OmpF mutants were expressed in the outer membrane of a porin deficient strain, E. coli BZB1107 from the plasmid pMS119 encoding the desired mutant ompF gene. Transformed cells were grown at 37ºC in LB (Luria-Bertani) medium supplemented with 100 µg/ml ampicillin, 30 µg/ml kanamycin and 0.2% (v/v) glucose in order to inhibit the expression of another outer membrane protein, LamB. PhoE porin expression is suppressed by the phosphate present in the media. When the OD600 of the cell culture reached
0.6, IPTG (final concentration of 1 mM) was added to induce protein expression and grown for a further 3 h. Cells were harvested by centrifugation at 8,000 g for 10 min at 4 ºC.
2.4.8 Extraction of OmpF from the outer membrane
The cell pellet was resuspended in 20 mM sodium phosphate, pH 6.8 and supplemented with DNase (10µg/ml) and RNase (10µg/ml). The cells were lysed by sonication of the cells on ice for 15 min. To remove unbroken cells
and cell debris, the suspension was centrifuged at 3,000 g for 15 min. After removing the pellet containing unbroken cells and cell debris, the supernatant was centrifuged at 40,000 rpm for 1 h in a 45-Ti rotor on a Beckman L7-80 ultracentrifuge in order to isolate the membrane pellet. The membrane pellet obtained was resuspended in wash buffer (20 mM Tris, 2% (w/v) SDS, pH 7.4) and then homogenised at 55ºC for 1 h. The solution was centrifuged at 40,000 rpm for 1 h in a 45-Ti rotor on a Beckman L7-80 ultracentrifuge and then the supernatant was discarded. The wash step was repeated twice. The extraction of OmpF from the remaining membrane pellet was carried out by incubating and homogenising the pellet in wash buffer with additional 500 mM NaCl. After incubation for 1 h at room temperature, the extract was centrifuged at 40,000 rpm for 1 h in a 45-Ti rotor on a Beckman L7-80 ultracentrifuge. The extraction was repeated once to increase the yield of OmpF. The supernatant containing SDS solubilised OmpF was dialysed against 5 mM NaHCO3, 0.1% (w/v) SDS
overnight at 37 ºC. The precipitation of OmpF (to concentrate or change detergent) was achieved by mixing cold ethanol, pre-chilled at -80 ºC, with OmpF samples to give a final ethanol concentration of 90% (v/v). This solution was then incubated at -20 ºC overnight. To isolate the precipitated OmpF pellet, the solution was centrifuged at 17,000 g for 1 h. The OmpF pellet was dried under a stream of air. OmpF was recovered by resuspension in 20 mM Tris- HCl, pH 7.5, 300 mM NaCl containing an appropriate detergent such as OG or octyl-POE.
2.4.9 Expression and isolation of OmpF inclusion bodies
OmpF inclusion bodies were expressed in E. coli BZB1107 from a pMS119 plasmid in which the ompF signal sequence (residues 1–22) plus the initial Ala23 residue is replaced by a single methionine residue to give a new start codon. Inclusion bodies were purified by BugBuster solution (Novagen) and solubilised with the denaturation buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, and 6 M guanidine HCl) and dialysed into 50 mM Bis-Tris, pH 7.0, 6 M urea at room temperature. Proteins extracted from inclusion bodies were purified using anion exchange chromatography in a 1-ml HiTrap Q Sepharose
2.4.10 Preparation of refolded OmpF
This was performed as in Visudtiphole et al with slight modification (Visudtiphole
et al., 2005). The purified OmpF from inclusion bodies was refolded by a 20x
dilution in 50 mM Tris-HCl, pH 8.0, 1 mM DTT (dithiothreitol) and 0.1 mM EDTA containing the mixture of 1% (w/v) DG (n-dodecyl-β-D-glucopyranoside) and 0.4% (w/v) DDM (n-dodecyl-β-D-maltoside). After a stationary incubation at 37ºC for at least 3 days, the sample was precipitated by mixing 1:9 with cold ethanol (see above) and then the refolded trimeric OmpF was recovered by resuspending in 20 mM Tris-HCl, pH 7.4, 0.5% (v/v) Octyl POE. To completely exchange the refolding detergents to another detergent, the buffer exchange was achieved by anion exchange chromatography using a 1-ml HiTrap Q Sepharose column equilibrated with 20 mM Tris-HCl, pH 7.4, 0.5% (v/v) octyl POE. The refolded OmpF were eluted using a salt gradient.