2. M ATERIAL AND M ETHODS
2.2 Methods
2.2.7 Work with proteins
2.2.7 Work with proteins
Overproduction of recombinant proteins in E.coli and cell disruption by French press For overexpression of Strep-tagged proteins in E. coli BL21, 500 ml LB + ampicillin were inoculated to an OD600 of 0.1 using an overnight culture of the respective E.coli strain carrying the overexpression plasmid. The culture was shaken at 37°C until an OD600 of 0.6-0.8 was reached and the overexpression was induced using 1mM IPTG. After 2.5 h of induction, the culture was harvested by centrifugation at 5,000 rpm for 20 min and 4°C. The pellet was resuspended in 15 ml buffer W and centrifuged in a falcon tube for 15 min at 4°C and 8,500 rpm. The supernatant was discarded and the pellet was used for cell disruption or stored at -20°C until further use.
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The disruption of E. coli cells after protein overexpression was performed using the SLM Aminco 2-FA-078-E1 French Press Cell (SLM Aminco) at 18000 psi and repeated 2 times. The disrupted suspension was centrifuged for 20 min at 4°C and 8,500 rpm to remove the major cell debris.
Afterwards, the supernatant was centrifuged for an additional hour at 35,000 rpm and 4°C in an ultracentrifuge. The pellet was discarded and the supernatant containing the soluble proteins was used as crude extract for purification.
Purification of proteins via Strep-Tactin® sepharose column
The proteins that were supposed to be purified were tagged with a Strep-tag II, an eight amino acids short peptide (WSHPQFEK), and purified using a Strep-Tactin® sepharose column (IBA) with a matrix volume of 0.5 ml. This Strep-Tactin® matrix binds specifically and with high affinity to the Strep-tag II of the desired proteins thereby separating them from the mixed protein solution. The column was equilibrated with 5 ml buffer W and loaded with the crude extract. The flow through was collected and the column was washed 4 times with 1.25 ml buffer W. For elution of the bound protein, initially 0.25 ml followed by three times 0.5 ml of buffer E were added. The elution fractions (E1-E4) as well as the last washing fraction (W4), the crude extract (CE) and the flow through (FT) were analyzed on a 12%
SDS-PAA-gel.
Buffer W 100 mM Tris-HCl pH 8.0
150 mM NaCl
1 mM EDTA
Buffer E 100 mM Tris-HCl pH 8.0
150 mM NaCl
1 mM EDTA
2.5 mM Desthiobiotin
Denaturing polyacrylamide gel electrophoresis (SDS-PAGE)
The denaturing gels were prepared according to Laemmli et al., 1970. These gels are poured to a thickness of 1 mm and consist of a stacking gel to accumulate the proteins in the sample and a running gel to separate the proteins according to their size. The samples were mixed with SDS-Loading dye (2 x) and boiled at 95°C for 5-10 min. After applying the samples, the gels were run at 120 V. In case of the Hoefer-SE-400 device, the gels were run over night at 30 V.
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Stacking gel 1.3 ml Rotiphorese® Gel 30 (37.5:1) 0.87 ml Tris-HCl pH 6.8 (1.5 M)
50 µl SDS (20%)
100 µl APS (10%)
20 µl TEMED
6.3 ml dH2O
Running gel (12%) 4 ml Rotiphorese® Gel 30 (37.5:1)
2.6 ml Tris-HCl pH 8.8 (1.5 M)
50 µl SDS (20%)
100 µl APS (10%)
10 µl TEMED
3.3 ml dH2O
5 x SDS loading dye 1.4 ml Tris-HCl 1 M pH 7.0
3 ml Glycerol
2 ml SDS (20%)
1.6 ml β-Mercaptoethanol 10 mg Bromophenol blue
2 ml dH2O
Running buffer (10 x) 1.92 M Glycerin
0.5 M Tris
10% SDS
Coomassie staining of polyacrylamide gels
After the gel run, the PAA gel was incubated for 15 min in fixing solution, stained with Coomassie staining solution for 5-10 min and destained until an optimal contrast between protein bands and background was observable. All steps were performed at room temperature with gentle shaking.
Fixing solution 500 ml Methanol
100 ml Acetic acid ad 1 l dH2O
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Staining solution 2.5 g Coomassie brilliant blue R250 100 ml Acetic acid
500 ml Methanol
ad 1 l dH2O
Destaining solution 150 ml Acetic acid
100 ml Methanol ad 1 l dH2O
Silver staining of polyacrylamide gels
The silver staining of protein bands in SDS-gels was performed according to the method of Nesterenko (1994). This was done by incubating the gels at room temperature and with gentle shaking in the following solutions:
Step Reagent Duration
Fixing Fixing solution 1 -24 h
Washing 50% ethanol 3 x 20 min
Reduction Thiosulfate solution 1 min
Washing dH2O 3 x 20 s
Staining Impregnating solution 25 min
Washing dH2O 2 x 20 s
Developing Developer Until sufficiently stained
Washing dH2O 5 s
Stopping Stop solution 5 min
Fixing solution 50 ml Methanol
12 ml Acetic acid
100 μl Formaldehyde (37%) ad 100 ml dH2O
Thiosulfate solution 20 mg Na2S2O3 x 5 H2O ad 100 ml dH2O
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Impregnating solution 0.2 g AgNO3
37 μl Formaldehyde (37%) ad 100 ml dH2O
Developer 6 g Na2CO3
2 ml Thiosulfate solution 50 μl Formaldehyde (37%) ad 100 ml dH2O
Stop solution 1.86 g EDTA
ad 100 ml dH2O
Western Blot
The Western blot was employed to detect Strep-tagged proteins from an SDS gel using an anti-Strep antibody (PromoKine) after protein purification. Using a semi-dry blotting apparatus (Peqlab), the proteins were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad).
At first, three Whatman papers were soaked in transfer buffer and placed onto the transfer device. The membrane was briefly activated in 100% methanol, equilibrated in transfer buffer and placed on top of the Whatman papers. The acrylamide gel was equilibrated in transfer buffer and placed onto the PVDF membrane. Finally, three Whatman papers soaked in transfer buffer were placed on the polyacrylamide gel. Bubbles were removed, the transfer device was closed and a current of 0.8 mA/cm2 was applied for 1 to 1.5 h. After the transfer of the proteins to the membrane, the membrane was incubated 1xTBS for 2 h. Subsequently, the membrane was incubated with the primary antibody (anti-Strep-antibody) overnight at 4°C. The anti-Strep-antibody was diluted 1:1,000 in TBS-tween.
Afterwards, the primary antibody was removed and three washing steps of 30 min in blocking solution followed. The membrane was incubated for 30 min with the secondary antibody, a polyclonal goat anti-rabbit immunoglobulin conjugated with alkaline phosphatase which was diluted 1:100,000 with blocking solution. The membrane was subsequently rinsed with deionized water and incubated in buffer III for 5 min. The membrane was placed between a transparent foil and the membrane was covered with a mixture of 500 μl buffer III and 5 μl CDP*. The luminescence was detected with a Chemolumineszenz Imager (modified from Rempeters, 2011).
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Blocking solution 100 ml 10 x TBS
25 g Milk powder
1 ml Tween® 20
ad 1 l dH2O
Buffer III (1 l) 0.1 M Tris
0.1 M NaCl
ad 1 l dH2O adjust pH 9.5
10 x TBS (1 l) 60 g Tris-HCl pH 7.6
90 g NaCl
ad 1 l dH2O
Transfer buffer (5 l) 15.1 g Tris
72.1 g Glycerol 750 ml Methanol ad 5 l dH2O
Preparation of M. pneumoniae protein extracts
The cells of a 100 ml culture were harvested after four days of growth in 1.5 ml PBS pH 7.4 in a 2 ml Eppendorf reaction tube. The cells were centrifuged (10 min, 11,000 x g, 4°C), washed three times and resuspended in 500 µl PBS. The cell suspension was transferred into a 2 ml screw-cap reaction tube containing 0.5 g glass beads (0.1 mm diameter). The cells and beads were vortexed 10 times for 30 s at 1 min intervals and subsequently centrifuged. The resulting supernatant contained the protein extract of the lysed M. pneumoniae cells.