Team:Bielefeld-Germany/Protocols/Downstream-processing

From 2011.igem.org

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{{Bielefeld_2011_Header}}
{{Bielefeld_2011_Header}}
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<html><img src="https://static.igem.org/mediawiki/2011/0/0f/Bielefeld-header-protocols-production.png"/><p></p></html>
'''Production Protocols:''' These are the protocols for the cultivation and the downstream processing.  
'''Production Protocols:''' These are the protocols for the cultivation and the downstream processing.  
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==Cultivation==
==Cultivation==
-
===Expression of [http://expasy.org/sprot/hamap/CORGL.html ''Brevibacterium flavum''] and [http://ijs.sgmjournals.org/cgi/content/abstract/54/3/779 ''Corynebacterium halotolerans''] S-layer genes===
+
===Expression of S-layer genes in ''E. coli''===
 +
<!-- * Used BioBricks: <partinfo>K525131</partinfo>, <partinfo>K525133</partinfo>, <partinfo>K525231</partinfo>, <partinfo>K525232</partinfo>, <partinfo>K525233</partinfo>, <partinfo>K525234</partinfo>, <partinfo>K525304</partinfo>, <partinfo>K525305</partinfo>, <partinfo>K525306</partinfo>, <partinfo>K525406</partinfo>, <partinfo>K525323</partinfo> -->
 +
* Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX]
 +
* Medium: [[Team:Bielefeld-Germany/Protocols/Materials#LB_medium | LB medium]] supplemented with 20 mg L<sup>-1</sup> chloramphenicol or [[Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium_for_KRX | autoinduction medium]]
 +
** Cultivations in LB-medium were supplemented with 0.1 % [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Used_chemicals L-rhamnose] as inducer, when the designated OD<sub>600</sub> was reached.
 +
** Autoinduction medium for expressing <partinfo>K525304</partinfo>, <partinfo>K525305</partinfo>, <partinfo>K525306</partinfo>, <partinfo>K525405</partinfo>, <!-- <partinfo>K525323</partinfo> --> was supplemented with 1 mM [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Used_chemicals IPTG].
 +
For characterising the expression rate and the influance on ''E. coli'' growth behavior an automatic sampling system (Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath BU) was used. These cultivations were carried out in an Infors AG AQUATRON.
 +
* 150 mL culture in 500 mL shaking flask with baffles (Schott) with silicon plugs
 +
* Cultivation temperature: 37 °C at 120 rpm
 +
 
===Expression of bisphenol A degrading BioBricks in ''E. coli''===
===Expression of bisphenol A degrading BioBricks in ''E. coli''===
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</div>
</div>
</html>
</html>
-
 
+
* Used BioBricks: <!-- <partinfo>K525502</partinfo> -->, <partinfo>K525512</partinfo>, <partinfo>K525517</partinfo>, <partinfo>K525552</partinfo>
-
* Used BioBricks: <partinfo>K525502</partinfo>, <partinfo>K525512</partinfo>, <partinfo>K525517</partinfo>, <partinfo>K525552</partinfo>
+
-
 
+
* Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX]
* Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX]
-
 
+
* Medium: [[Team:Bielefeld-Germany/Protocols/Materials#LB_medium | LB medium]] supplemented with 100 mg L<sup>-1</sup> ampicillin and 120 mg L<sup>-1</sup> [[Team:Bielefeld-Germany/Protocols/Materials#Used_chemicals | bisphenol A]]
-
* Medium: [[Team:Bielefeld-Germany/Protocols#LB_medium | LB medium]] supplemented with 100 mg L<sup>-1</sup> Ampicillin and 120 mg L<sup>-1</sup> [[Team:Bielefeld-Germany/Protocols#Used_chemicals | bisphenol A]]
+
** BPA is thermally stable -> you can autoclave it together with the medium
** BPA is thermally stable -> you can autoclave it together with the medium
-
 
* 100 mL culture in 300 mL shaking flask without baffles (Schott) with silicon plugs
* 100 mL culture in 300 mL shaking flask without baffles (Schott) with silicon plugs
-
 
* Cultivation temperature: 24 °C, 30 °C or 37 °C, tempered with Infors AG AQUATRON at 120 rpm
* Cultivation temperature: 24 °C, 30 °C or 37 °C, tempered with Infors AG AQUATRON at 120 rpm
-
 
* for characterizations: automatic sampling every three hours with Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath <html><a href="http://www.abrf.org/index.cfm/list.msg/abrf/23205" style="color:white">BURMA-SHAVE!!</a></html>
* for characterizations: automatic sampling every three hours with Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath <html><a href="http://www.abrf.org/index.cfm/list.msg/abrf/23205" style="color:white">BURMA-SHAVE!!</a></html>
** the characterization experiment setup is shown on the picture on the right
** the characterization experiment setup is shown on the picture on the right
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===Bioreactor cultivations with ''E. coli'' KRX===
===Bioreactor cultivations with ''E. coli'' KRX===
-
 
+
To obtain higher amounts and concentration of proteins we cultivated and expressed in a bioreactor. It is possible to cultivate several liters and to control temperature, pH and DO.
-
To obtain higher amounts and concentration of proteins we cultivated and expressed in a bioreactor.  
+
* Bioreactor: Bioengineering NLF22 7 L or [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with Bioengineering DCU
-
 
+
* Medium: [[Team:Bielefeld-Germany/Protocols/Materials#HSG_medium | HSG medium]] with 20 mg L<sup>-1</sup> chloramphenicol or 100 mg L<sup>-1</sup> ampicillin
-
 
+
* Culture volume: 2.5 - 5 L
-
* Bioreactor: [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU
+
* Starting OD<sub>600</sub>: 0.1 - 0.4
-
 
+
-
* Medium: HSG medium with 20 mg mL<sup>-1</sup> chloramphenicol
+
-
 
+
-
* Culture volume: 4 L
+
-
 
+
* DO: 40 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
* DO: 40 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
-
 
* pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
* pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
 +
* Antifoam: BASF pluronic PE-8100
 +
* Induction after 4 h cultivation time with 0.2 % rhamnose and 1 mM IPTG (in culture medium)
 +
* Harvest after 8 - 13 h
-
* Antifoam: BASF pluronic XXX
 
-
* Induction after 4 h cultivation time with 2 % rhamnose (in culture medium)
+
==Purification methods==
-
==Purification methods==
+
===Enzymatic cell lysis with lysozyme===
 +
* After cultivation biomass was collected by centrifugation at 5,000 ''g'' at 4 °C for 20 min.
 +
* 1 g of biomass (wet weight) was suspended in 10 mL of enzyme buffer containing 0.1 % Triton X-100, 2 µL benzonase (250 U/µL) and 40 µL of lysozyme (100 mg mL<sup>-1</sup>)
 +
* Incubation for 30 min at 4 °C
 +
* reaction mixture was centrifuged for 30 - 90 min at 15,000 ''g'' at 4 °C
===Release of periplasmic protein fraction from ''E. coli'' by cold osmotic shock===
===Release of periplasmic protein fraction from ''E. coli'' by cold osmotic shock===
-
 
Modified protocol from [http://www.jbc.org/content/240/9/3685.full.pdf+html?sid=4a90c176-0ec3-489f-8c82-4734274cebf5 Neu & Heppel, 1965].
Modified protocol from [http://www.jbc.org/content/240/9/3685.full.pdf+html?sid=4a90c176-0ec3-489f-8c82-4734274cebf5 Neu & Heppel, 1965].
-
 
+
* Centrifuge ''E. coli'' cell suspension for 5 min at 14,000 ''g'' (4 °C) to collect the cells.
-
* Centrifuge ''E. coli'' cell suspension for 5 min at 14000 ''g'' (4 °C) to collect the cells.
+
* Discard the entire supernatant.
* Discard the entire supernatant.
-
* Resuspend the cells ice-cold [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Cold_osmotic_shock_buffers_for_the_release_of_periplasmic_protein_fraction cell fractionating buffer #1]. The resulting volume should be 1/4 of the former suspension volume.
+
* Resuspend the cells ice-cold [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Cold_osmotic_shock_buffers_for_the_release_of_periplasmic_protein_fraction cell fractionating buffer 1]. The resulting volume should be 1/4 of the former suspension volume.
* Incubate for 20 min on ice. Ivert the suspension at regular intervals to counteract sedimentation.
* Incubate for 20 min on ice. Ivert the suspension at regular intervals to counteract sedimentation.
-
* Centrifuge the cell suspension for 15 min at 14000 g (4 °C).
+
* Centrifuge the cell suspension for 15 min at 14,000 g (4 °C).
* Discard the entire supernatant.
* Discard the entire supernatant.
-
* Resuspend the cells ice-cold [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Cold_osmotic_shock_buffers_for_the_release_of_periplasmic_protein_fraction cell fractionating buffer #2]. The resulting volume should be 1/4 of the former suspension volume.
+
* Resuspend the cells ice-cold [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Cold_osmotic_shock_buffers_for_the_release_of_periplasmic_protein_fraction cell fractionating buffer 2]. The resulting volume should be 1/4 of the former suspension volume.
* Incubate for 10 min on ice under regular invertion.
* Incubate for 10 min on ice under regular invertion.
-
* Centrifuge the cell suspension for 15 min at 14000 g (4 °C).
+
* Centrifuge the cell suspension for 15 min at 14,000 g (4 °C).
* Save the supernatant, which contains the periplasmatic proteins.
* Save the supernatant, which contains the periplasmatic proteins.
* If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter.
* If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter.
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===Inclusion body clean-up===
===Inclusion body clean-up===
-
 
* harvest the cells by centrifugation (30 min, 10,000 g, 4 °C)
* harvest the cells by centrifugation (30 min, 10,000 g, 4 °C)
* resuspend pellet and disrupt cells
* resuspend pellet and disrupt cells
-
* centrifuge lysate (60 min, >17,000 g, 4 °C)
+
* centrifuge lysate (60 - 90 min, >17000 g, 4 °C)
* wash pellet at least two times with water to remove water-soluble proteins
* wash pellet at least two times with water to remove water-soluble proteins
-
* after washing the pellet: incubate the pellet in [[Team:Bielefeld-Germany/Protocols#Denaturation_buffer_for_inclusion_bodies | denaturation buffer]] for 60 min, 4 °C with vertical rotator
+
* after washing the pellet: incubate the pellet in [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Denaturation_buffer_for_inclusion_bodies denaturation buffer] for 60 min, 4 °C with vertical rotator
** final concentration in denaturation buffer: 0.5 mg wet biomass per mL
** final concentration in denaturation buffer: 0.5 mg wet biomass per mL
* centrifuge (60 min, >17,000 g, 4 °C)
* centrifuge (60 min, >17,000 g, 4 °C)
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* centrifuge (60 min, >17,000 g, 4 °C)
* centrifuge (60 min, >17,000 g, 4 °C)
* collect supernatant and discard pellet
* collect supernatant and discard pellet
 +
 +
 +
===Ammonium sulfate precipitation===
 +
* Mix fraction you want to clean-up with ammonium sulfate
 +
** To precipitate S-layer proteins from ''Corynebacterium'', 40 % ammonium sulfate saturation concentration is a good concentration (247 g L<sup>-1</sup> ammonium sulfate at 25 °C)
 +
* Incubate 30 min at room temperature on a shaker
 +
* Centrifuge (the faster and longer the better) and solve the precipitate in water or buffer
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</div>
</div>
</html>
</html>
 +
* Arrange the filtration module as shown on the right side.
 +
* Microfiltration (0.22 µm) or cross flow filtration with 300 kDa (we used a Milipore Pellicon XL 300) membrane of sample before ultrafiltration.
 +
* For concentrating the sample just filter it until the desired volume is left in the feed reservoir. For diafiltration (e.g. buffer exchange, desalting) dilute the feed reservoir several times and filter continously.
 +
* Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
 +
** 50 or 100 kDa cut-off
 +
** 50 cm<sup>2</sup> filtration area
 +
** tangential flow filter
 +
** Hydrophilic polyvinylidene fluoride membrane
 +
* Used pump: SciLog TANDEM 1081 peristaltic pump
 +
** flow rate during filtration: 40 mL min<sup>-1</sup>
-
* Milipore ... XL 50 and 100 kDa membranes
 
-
 
-
* pump
 
-
 
-
* bla bla
 
===Ion exchange chromatography (IEX) for S-layer proteins from ''Corynebacterium''===
===Ion exchange chromatography (IEX) for S-layer proteins from ''Corynebacterium''===
-
 
+
* used column: DEAE HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
-
* used column: DEAE HiTrap 1 mL with GE Healthcare ÄKTA
+
* flow rate: 1 mL min<sup>-1</sup>
* flow rate: 1 mL min<sup>-1</sup>
-
 
+
* equilibrate column with > 10 column volumes of [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX | binding buffer]]
-
* equilibrate column with 5 column volumes of [[Team:Bielefeld-Germany/Protocols#Buffers_for_S-layer_IEX | binding buffer]]
+
* inject sample and wash column with binding buffer until UV signal is constant
-
* inject sample and wash column with 5 column volumes of binding buffer
+
* elute with 20 %, 40 % and 60 % of [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX | binding / elution buffer]] mix and collect fractions
-
* elute S-layer with 40 % [[Team:Bielefeld-Germany/Protocols#Buffers_for_S-layer_IEX | binding / elution buffer]] mix
+
* elute remaining proteins with 100 % elution buffer
* elute remaining proteins with 100 % elution buffer
 +
===Ion exchange chromatography (IEX) for S-layer proteins from ''Lysinibacillus sphaericus''===
 +
* tested with <partinfo>K525405</partinfo>
 +
* used column: DEAE HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
 +
* flow rate: 0.5 mL min<sup>-1</sup>
 +
* equilibrate column with 20 column volumes of [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX | binding buffer]]
 +
* inject sample and wash column with binding buffer until UV signal is constant
 +
* elute with 10 % of [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX | binding / elution buffer]] mix and collect fraction
 +
* elute remaining proteins with 100 % elution buffer
-
===Ni-NTA spin columns===
 
-
denaturating
+
===Hydrophobic Interaction Chromatography (HIC) for S-layer proteins===
-
 
+
* tested with <partinfo>K525405</partinfo> and <partinfo>K525311</partinfo>
-
non-denaturating
+
* used column: Butyl HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
 +
* flow rate: 0.5 mL min<sup>-1</sup>
 +
* equilibrate column with 20 column volumes of [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_HIC | binding buffer]]
 +
* inject sample and wash column with binding buffer
 +
* elute in 10 % steps of [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_HIC | binding / elution buffer]] mix and collect fractions
 +
** <partinfo>K525405</partinfo> elutes at 70 % buffer B
 +
** <partinfo>K525311</partinfo> elutes at 50 % buffer B
 +
* elute remaining proteins with 100 % elution buffer
===His-tag affinity chromatography===
===His-tag affinity chromatography===
 +
*Column: 1&nbsp;mL HisTrap FF crude by [http://www.gehealthcare.com/ GE Healthcare]
 +
*Harvest cells by centrifugation at 10,000&nbsp;g for 10&nbsp;min at 4&nbsp;°C
 +
*Discard the supernatant and freeze bacterial pellet at -20&nbsp;°C for at least 30&nbsp;min
 +
*Resuspend the pellet in 5&nbsp;mL binding buffer for each gram of cell paste
 +
*Wash column with 5 - 10&nbsp;mL of deionized water
 +
*Equilibrate column with 5 - 10&nbsp;mL of binding buffer
-
==Bradford Protein Assay==
+
'''denaturing'''
 +
*For buffers see table [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography  buffers for his-tag affinity chromatography]
 +
*Mechanical lysis:
 +
**Sonification on ice for 6 - 10 min with Sonifier 450 by [http://www.gehealthcare.com/ Branson], max. 20 W, cooled on ice
 +
*Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
 +
*Centrifuge at 15,000&nbsp;g for 30&nbsp;min at 4&nbsp;°C
 +
*Filter sample (sterile filter or 300 kDa cut-off)
-
==Measuring of [http://partsregistry.org/Part:BBa_E1010 mRFP]==
 
-
* Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
 
-
* Freeze samples at -80 °C for storage
 
-
* To measure the samples thaw at room temperature and fill 200 µL of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)
 
-
* Measure the fluorescence in a platereader (we used a [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]) with following settings:
 
-
** 20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm)
 
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** Measurement mode: Top
 
-
** Excitation: 584 nm
 
-
** Emission: 620 nm
 
-
** Number of reads: 25
 
-
** Manual gain: 100
 
-
** Integration time: 20 µs
 
-
==Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)==
+
*Syringe method
 +
**Equilibrate with binding buffer
 +
**Load sample onto column
 +
**Wash with 10&nbsp;mL binding buffer
 +
**Elute with 5&nbsp;mL of elution buffer with increasing imidazole concentrations
 +
**Collect the eluate in 1&nbsp;mL fractions, the purified protein is most likely in the second or third fraction
 +
**Re-equilibrate the column with binding buffer
-
This analytical Method can be used for separation and identification of proteins according to their electrophoretic mobility. The mobility is a function of length of the molecular weight. Proteins that have identical charge per unit mass due to binding of SDS results in an equal electrophoretic mobility.
 
-
===Pouring the polyacrylamide gel===
+
*ÄKTA method
 +
**Equilibrate with 20 column volumes binding buffer, 0.5 mL min<sup>-1</sup>
 +
**Load sample onto column
 +
**Wash with binding buffer until UV signal is constant
 +
**Elute with 50 mM imidazol
 +
**Elute remaining proteins with 500 mM imidazol
 +
-
*Make a master mix for the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#SDS-PAGE_gel stacking] and [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#SDS-PAGE_gel separating gel] without adding ammonium persulfate and TEMED.
+
'''non-denaturing'''
-
*Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel.
+
*For buffers see table [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography  buffers for his-tag affinity chromatography]
-
*Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel.
+
*Enzymatic lysis:
-
*Layer isopropanol on top of the gel.
+
**Add 0.2&nbsp;mg&nbsp;L<sup>-1</sup> lysozyme, 3 units of Benzonase per mL of culture volume and 1&nbsp;mM MgCl<sub>2</sub>
-
*Leave the separating gel at room temperature for >60 minutes to polymerize.
+
**Stirr for 30&nbsp;in at 4&nbsp;°C
-
*Remove isopropanol and wait until the surface is dry.
+
*Centrifuge at 10,000&nbsp;g for 30&nbsp;min at 4&nbsp;°C
-
*Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form.
+
*Load sample onto the column
-
*Insert comb without getting bubbles stuck underneath
+
*Wash with 10&nbsp;mL binding buffer
-
*Leave the gel at room temperature for >60 minutes to polymerize.
+
*Elute with 5&nbsp;mL of elution buffer with increasing imidazole concentrations
 +
*Collect the eluate in 1&nbsp;mL fractions, the purified protein is most likely in the second or third fraction
 +
*Re-equilibrate the column with binding buffer
-
*For storage
 
-
**Remove sealing and store the gel wrapped in moistened paper towel at 4°C.
 
-
===Preparing the sample===
+
==Production and purification strategies==
-
*Mix your protein mixture 4:1 with [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#4x_Laemmli-buffer Laemmli-buffer] (30 mL protein solution + 10 mL Laemmli-buffer)
+
===Fusion proteins of SgsE and SbpA===
-
*Heat for 5 minutes at 95 °C.
+
* Cultivation
 +
** Bioreactor: Bioengineering NLF22 7 L or [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] with Bioengineering DCU
 +
** Medium: [[Team:Bielefeld-Germany/Protocols/Materials#HSG_medium | HSG medium]] with 20 mg L<sup>-1</sup> chloramphenicol or 100 mg L<sup>-1</sup>
 +
** Culture volume: 2.5 - 5 L
 +
** Inoculation OD<sub>600</sub>: 0.1 - 0.4
 +
* DO: 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
 +
* pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
 +
* Antifoam: BASF pluronic PE-8100
 +
* Induction after 4 h cultivation time with 0.2 % rhamnose and 1 mM IPTG (in culture medium)
 +
* Harvest after 8 - 13 h
-
===Running the gel===
+
*Cell lysis
 +
** Centrifuge down the cells (10,000 g, 30 min, 4 °C)
 +
** Resuspend pellet in enzyme buffer or binding buffer for denaturing his-tag purification
 +
** Cell lysis with high-pressure homogenizer (800 bar, 3 cycles at 4 °C)
 +
** Centrifuge down the lysate (10,000 g, 60 min, 4 °C)
-
*Remove sealing, put the polymerized gel into gel box and pour [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#SDS_running_buffer SDS running buffer] into the negative and positive electrode chamber.
+
*Inclusion body clean-up
-
*Remove comp without destroying the gel pocket.
+
** wash pellet from cell lysis with water twice
-
*Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample.
+
** after washing the pellet: incubate the pellet in [[Team:Bielefeld-Germany/Protocols/Materials#Denaturation_buffer_for_inclusion_bodies | denaturation buffer]] for 60 min, 4 °C with vertical rotator
-
*Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel.
+
*** final concentration in denaturation buffer: 0.5 mg wet biomass per mL
-
*Raise amperage up to 20 mA for running the separating gel.
+
** centrifuge (60 min, >17,000 g, 4 °C)
-
*When the distance of the lowest molecular weight standard lane to the gel end is down to 0,5 cm stop the electrophoresis by turning off the power supply.
+
*** in general with all centrifugations during this clean-up: the higher the speed, the better the result
 +
** collect supernatant and incubate the pellet again in denaturation buffer (60 min, 4 °C, vertical rotator)
 +
** centrifuge (60 min, >17,000 g, 4 °C)
 +
** collect supernatant and discard pellet
 +
<html>
 +
<div style="float:right; width:400px; text-align:center;">
 +
<img src="https://static.igem.org/mediawiki/igem.org/5/5f/Bielefeld-Germany2011-filtrationmodule.jpeg" width="90%" height="90%" />
 +
<br/>
 +
</div>
 +
</html>
 +
*Filtration
 +
** Arrange the filtration module as shown on the right side.  
 +
** Collect permeate of cross flow filtration with 300 kDa membrane of sample before ultrafiltration
 +
*** This step is for removing cell debris
 +
** Diafiltrate with 100 kDa membrane against [[Team:Bielefeld-Germany/Protocols/Materials#Denaturation_buffer_for_inclusion_bodies | denaturation buffer]]
 +
*** constantly delute permeate with the buffer, keeping the permeate volume as low as possible
 +
** Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
 +
*** 50, 100 or 300 kDa cut-off
 +
*** 50 cm<sup>2</sup> filtration area
 +
*** tangential flow filter
 +
*** Hydrophilic polyvinylidene fluoride membrane
 +
** Used pump: SciLog TANDEM 1081 peristaltic pump
 +
*** flow rate during filtration: 40 mL min<sup>-1</sup>
-
==Polyacrylamide gel staining==
+
OR WHEN USING A HIS-TAGGED PROTEIN (RECOMMENDED):
-
===Colloidal Coomassie Blue staining===
+
*His-6-affinity tag purification
-
Modified staining protocol from [http://http://newjournal.kcsnet.or.kr/main/j_search/j_abstract_view.htm?code=B021105&qpage=j_search&spage=b_bkcs&dpage=ar Kang ''et al''., 2002].
+
**For buffers see table [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography  buffers for his-tag affinity chromatography]
 +
**Mechanical lysis as described above in binding buffer
 +
**Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
 +
**Centrifuge at 15,000&nbsp;g for 30&nbsp;min at 4&nbsp;°C
 +
**Filter sample (sterile filter or 300 kDa cut-off)
 +
**Equilibrate column (1 mL HisTrap FF crude from GE Healthcare) with 20 column volumes binding buffer, 0.5 mL min<sup>-1</sup>
 +
**Load sample onto column
 +
**Wash with binding buffer until UV signal is constant
 +
**Elute with 50 mM imidazol
 +
**Elute remaining proteins with 500 mM imidazol
-
*agitate the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Colloidal_Coomassie_Brilliant_Blue_G-250_staining_solution staining solution] at 37 °C over night to form the colloids
+
* Dialysis
-
*After finishing the SDS-PAGE remove gel from gel casting form and tranfer it in to a box.
+
** Fill retentate from DF/UF or chromatography elution fraction in dialysis tube ([http://www.carl-roth.de/ Roth], cellulose, 10 kDa cut-off)
-
*Add 100 mL of the stainig solution to your polyacrylamid gel.
+
** Dialyse against ddH<sub>2</sub>O for 18 h at 4 °C in the dark
-
*Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation.
+
** After dialysis: centrifuge down the precipitation (45 min, 17,000 g, 4 °C) and collect the supernatant
-
*Remove the staining solution
+
** Measure protein concentration in supernatant, dilute to 1 mg mL<sup>-1</sup> with ddH<sub>2</sub>O and store at 4 °C in the dark
-
*wash the gel with dH<sub>2</sub>O
+
-
*Incubate the gel in ddH<sub>2</sub>O (2-6h) for bleaching the background. Shake the gel continuously during incubation.  If necessary replace the colored water with new one.
+
-
===Silver staining===
+
* Scheme of purification strategy for S-layer (fusion) proteins:
-
Modified staining protocol from
+
With inclusion body purification:
-
*After finishing the SDS-PAGE remove gel from gel casting form and tranfer it in to a box.
+
[[Image:Bielefeld-Germany2011-305_405-Aufreinigung_symbol.png|800px|center]]
-
*Add 50 mL of fixation solution and incubate the gel 1 to 24 h. The formaldehyde (37 %) must be added to the solution short time before.
+
-
*Remove the fixation solution and wash the gel 3 times (1-20 min) with  50 mL of 50 % (v/v) ethanol.
+
-
*Add 50 mL of thiosulfate solution and incubate the gel exactly for 1 min. If the gel incubates to long the background  becomes to dark.
+
-
*Remove thiosulfate solution and wash the gel 3 times (20 s) with dH<sub>2</sub>O.
+
-
*Add 50 mL of impregnation solution and icubate the gel for 15 to 20 min. The formaldehyde (37 %) must be added to the solution short time before.
+
-
*Remove impregnation solution and wash the gel 2 times (20 s) with dH<sub>2</sub>O.
+
-
*Add 50 mL of developing solution and incubate the gel until protein bands become visible.
+
-
*Remove the developing solution immediately and wash the gel for 10 to 20 s with dH<sub>2</sub>O.
+
-
*Add 50 mL of stop solution and incubate the gel 10 to 20 s.
+
-
*Remove the stop solution and wash the gel for 1 min with dH<sub>2</sub>O.
+
-
==Tryptic digest of gel lanes for analysis with MALDI-TOF==
+
With HisTrap:
 +
[[Image:IGEM-Bielefeld2011-322_Aufreinigung_symbol.png|800px|center]]
-
Note:
+
* Further purification of dirty inclusion body purification by IEX and HIC as described above
-
*Make sure to work under a fume hood.
+
-
*Do not work with protective gloves to prevent contamination of your sample with platicizers.
+
-
Reaction tubes have to be cleaned with 60% (v/v) CH<sub>3</sub>CN, 0,1% (v/v) TFA. Afterwards the solution has to be removed completely followed by evaporation of the tubes under a fume hood. Alternatively microtiter plates from Greiner® (REF 650161) can be used without washing.
 
-
*Cut out the protein lanes of a Coomassie-stained SDS-PAGE using a clean scalpel. Gel parts are transferred to the washed reaction tubes/microtiter plate. If necessary cut the parts to smaller slices.
+
===Fusion proteins of CspB without lipid anchor with TAT-sequence===
-
*Gel slices should be washed two times. Therefore add 200 µL 30% (v/v) acetonitrile in 0,1 M ammonium hydrogen carbonate each time and shake lightly for 10 minutes. Remove supernatant and discard to special waste.
+
* Cultivation
-
*Dry gel slices at least 30 minutes in a Speedvac.
+
** 150 mL culture in 500 mL or 300 mL culture in 1000 mL shaking flasks with baffles (Schott) with silicon plugs
-
*Rehydrate gel slices in 15 µL Trypsin-solution followed by short centrifugation.
+
** Medium: [[Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium_for_KRX | autoinduction medium]]
-
*Gel slices have to be incubated 30 minutes at room temperature, followed by incubation at 37 °C overnight.
+
** Cultivation temperature: 37 °C at 120 rpm
-
*Dry gel slices at least 30 minutes in a Speedvac.
+
-
*According to the size of the gel slice, add 5 – 20 µL 50% (v/v) ACN / 0,1% (v/v) TFA.
+
-
*Samples can be used for MALDI measurement or stored at -20 °C.
+
-
Trypsin-solution: 1 µL Trypsin + 14 µL 10 mM NH<sub>4</sub>HCO<sub>3</sub>
+
* Supernatant precipitation
-
*Therefore solubilize lyophilized Trypsin from Promega® in 200 µL of provided buffer and incubate for 15 minutes at 30 °C for activation. For further use it can be stored at -20 °C.
+
** Centrifuge down the cells (10000 g, 30 min, 4 °C) and collect the supernatant
-
 
+
** To precipitate S-layer proteins from ''Corynebacterium'', i.e. 40 % ammonium sulfate saturation concentration (247 g L<sup>-1</sup> ammonium sulfate at 25 °C)
-
 
+
** Incubate 30 min at room temperature on a shaker
-
==Bisphenol A analysis==
+
** Centrifuge (the faster and longer the better) and solve the precipitate in [[Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX | binding buffer]] for IEX
-
 
+
<html>
-
===Extraction with ethylacetate===
+
<div style="float:right; width:400px; text-align:center;">
-
* mix 100 µL culture supernatant with 100 µL internal standard ([[Team:Bielefeld-Germany/Protocols#Used_chemicals | bisphenol F]], 100 µg L<sup>-1</sup>)
+
<img src="https://static.igem.org/mediawiki/igem.org/5/5f/Bielefeld-Germany2011-filtrationmodule.jpeg" width="90%" height="90%" />
-
* add 200 µL [[Team:Bielefeld-Germany/Protocols#Used_chemicals | ethylacetate (HPLC grade)]] for extraction
+
<br/>
-
* vortex (30 s)
+
</div>
-
* centrifuge for phase separation (5 min, 5000 g)
+
</html>
-
* take a bit from upper phase and put it in a clean eppi
+
*Filtration
-
* SpeedVac at 40 °C to remove ethlyacetate
+
** Arrange the filtration module as shown on the right side.  
-
* solve remaining BPA in water (HPLC grade), vortex (30 s)
+
** Collect permeate of cross flow filtration with 300 kDa membrane of sample before ultrafiltration
-
* solubility of BPA in water only 300 mg L<sup>-1</sup>
+
*** This step is for removing cell debris
-
** for LC-MS analysis of BPA, 300 mg BPA L<sup>-1</sup> is definitely enough
+
** Diafiltrate with 50 kDa membrane against [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX binding buffer] for IEX
-
** if you want to detect or expect higher concentrations of BPA, solve it in an acetonitrile-water-mix
+
*** constantly delute permeate with the buffer, keeping the permeate volume as low as possible
-
 
+
** Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50], XL 100 or XL 300 membranes
-
===HPLC method===
+
*** 50, 100 or 300 kDa cut-off
-
* C18 reverse phase column
+
*** 50 cm<sup>2</sup> filtration area
-
* Isocratic method: 45 % [[Team:Bielefeld-Germany/Protocols#Used_chemicals | Acetonitrile]]
+
*** tangential flow filter
-
* Flow = 0.6 mL min<sup>-1</sup>
+
*** Hydrophilic polyvinylidene fluoride membrane
-
* UV-detection at 227 nm
+
** Used pump: SciLog TANDEM 1081 peristaltic pump
-
* Internal standard: 100 mg L<sup>-1</sup> [[Team:Bielefeld-Germany/Protocols#Used_chemicals | Bisphenol F]] (BPF)
+
*** flow rate during filtration: 40 mL min<sup>-1</sup>
-
* Column:
+
-
** Eurospher II 100-5 C18p by [http://www.knauer.net/ Knauer]
+
-
** Dimensions: 150 x 4.6 mm with precolumn
+
-
** Particle size: 5 µm
+
-
** Pore size: 100 Å
+
-
** Material: silica gel
+
-
* Software:
+
-
** Clarity (Version 3.0.5.505) by [http://www.dataapex.com/ Data Apex]
+
-
* Autosampler:
+
-
** Midas by [http://www.spark.nl/ Spark Holland]
+
-
** Tray cooling: 10 °C
+
-
* Pump:
+
-
** L-6200A Intelligent Pump by [http://www.hitachi.com/ Hitachi]
+
-
* UV-Detector:
+
-
** Series 1050 by [http://www.hp.com/ Hewlett Packard]
+
-
 
+
-
[[File:Bielefeld2011 BPA + Abbauprodukt-2.JPG|900px|left|thumb|Chromatogram obtained using the above described materials and methods. Three distinctive peaks are of interest: BPA at 7,3 min retention time, BPF at 5,8 min retention time and the degradation product of BPA at 4,6 min retention time.]]
+
-
 
+
-
==NAD<sup>+</sup> detection==
+
-
For the [http://www.molecular-beacons.com/MB_SC_design.html design of molecular beacons] different available tools may help you so that a correct functionality is ensured under defined assay conditions. All measurements were made in the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer].
+
-
 
+
-
===Characterisation of molecular beacons===
+
-
 
+
-
Preferentially, use a complementary oligonucleotide (target) as well as two enshortened oligonucleotides (split target) for [http://www.molecular-beacons.com/MB_SC_protocol.html#cap3 characterising the molecular beacon] in its closed and open state. In this way you can verify simultaneously the ineffectiveness of the split target to reach the molecular beacon`s open state.
+
-
 
+
-
* Optimal wavelengths:
+
-
** Determine the emission spectrum of your molecular beacon by initially using a literature value for the exctintion wavelength of the labeled fluorophore. For this add 10 µL of 1 µM molecular beacon in 200 µL [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer], mix thoroughly and wait 2 min until measuring the fluorescence with a spectrofluorometer at an defined temperature (optimally 37 °C) .
+
-
** Add 10 µL of 2 µM either target or split target and shake at least 6 min until measuring the fluorescence again (when equilibrium is reached).
+
-
** Repeat the procedure to determine the extinction spectrum using the self-calculated optimal emission wavelength for your fluorophore/quencher combination. For further fluorescence measurements use the extinction wavelength for which the difference between the molecular beacons's closed state and the open state signals is maximal.
+
-
 
+
-
* Thermal profile analysis:
+
-
** Prepare a 30 µL reaction mix composed of [https://2011.igem.org/Team:Bielefeld-Germany./Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer] and 1 µM molecular beacon. In a separate sample add 2 µM either target or split target.
+
-
** Use a spectrofluorometric thermal cylcer to dertemine the fluorescence as a function of temperature. Start at 80 °C and decrease the temperature stepwise in 1 °C intervals holding each temperature for 1 min and measuring the fluorescence at the end of each step.
+
-
* Signal-to-background ratio (S/B):
+
* Ion exchange chromatography
-
[[Image:Bielefeld-Germany-2011 MB S-B-ratio.png|200px|thumb|right|Signal-to-background ratio]]
+
** used column: DEAE HiTrap 1 mL with [http://www.gehealthcare.com/ GE Healthcare] ÄKTA
 +
** flow rate: 1 mL min<sup>-1</sup>
 +
** equilibrate column with 10 column volumes of [Team:Bielefeld-Germany/Protocols#Buffers_for_S-layer_IEX | binding buffer]
 +
** inject sample and wash column with binding buffer
 +
** elute with 20 %, 40 % and 60 % [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_S-layer_IEX binding / elution buffer] mix and collect samples -> take the cleanest fraction for further work
 +
** elute remaining proteins with 100 % elution buffer
-
**The approach is quite similar to the one that determines the optimal wavelengths which should be used to monitor the fluorescence of the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer] (F<sub>buffer</sub>) and the added molecular beacon (F<sub>closed</sub>) as well as the target (F<sub>open</sub>) or split target. Monitor the fluorescence at the optimal wavelengths until the equilibrium is reached each time before you add a new component.
+
* Scheme of purification strategy for CspB S-layer (fusion) proteins:
-
** The signal-to-background ratio can be calculated as shown on the right. Finally, compare the signal-to-background ratios after adding the target and split target to check whether the split target has an effect on the melting of the molecular beacon's hairpin structure.
+
[[Image:Bielefeld-Germany2011-CH4-Aufreinigung_symbol.png|800px|center]]
-
* Imaging:
 
-
** Prepare a 200 µL reaction mix composed of [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer] and 500 nM molecular beacon in a PCR tube. In a separate sample add 600 µM either target or split target. Excitate the samples by a UV transilluminator and take images preferably with a camera that can detect different colours.
 
-
===Purification of DNA ligase===
+
==Recrystallization of S-layer proteins==
-
The purification of the overexpressed NAD<sup>+</sup>-dependent DNA ligase gene ([https://2011.igem.org/Team:Bielefeld-Germany/ BBa_K525710]) in ''E. coli'' was performed under native conditions and Ni-NTA columns were used utilizing the recombinant protein`s C-terminal 6xHis-tag.
+
-
*Cultivation
+
===Recrystallization of SgsE and SbpA in solution===
-
**Prepare an overnight culture of [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX] carrying the plasmid with DNA ligase ([https://2011.igem.org/Team:Bielefeld-Germany/ BBa_K525710]) in 30 ml [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#LB_medium LB medium] containing 20 μg mL<sup>-1</sup> chloramphenicol at 37 °C.
+
* after purification: dialyse 18 h at 4 °C against ddH<sub>2</sub>O in the dark
-
**Dilute the overnight culture in 100 ml [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Autoinduction_medium_for_KRX autoinduction medium] (20 μg mL<sup>-1</sup> chloramphenicol added) to an OD<sub>600</sub> = 0,1 and harvest the cells after 4 h growth at 37 °C.
+
* centrifuge and use supernatant -> this is the monomeric protein solution
-
**Use the centrifuged cells immediately for protein purification or store them at -20 °C.
+
* dialyse 18 h at 4 °C against
 +
** [[Team:Bielefeld-Germany/Protocols/Materials#Hanks_Buffered_Saline_Solution_.28HBSS.29 | HBSS]] pH 7.4 for SgsE
 +
** recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl<sub>2</sub>) for SbpA
-
*Small-scale purification
 
-
**Resuspend a pellet derived from 5 ml cell culture volume in 630 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10].
 
-
**Add 70 μl lysozyme (10 mg/ml) as well as 25 Units benzonase nuclease and mix thoroughly.
 
-
**Incubate on ice for 30 min.
 
-
**Centrifuge the lysate for 30 min at 12000 g (4 °C). Collect the supernatant (cleared lysate).
 
-
**Prepare a [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 Ni-NTA spin column] ([http://www.qiagen.com/default.aspx QIAGEN]) by equilibration with 600 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10] and centrifugation for 2 min at 890 g (4 °C).
 
-
**Load up to 600 μl cleared lysate onto the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 Ni-NTA spin column] and centrifuge for 5 min at 270 g (4 °C). Collect the flow-through.
 
-
**Wash the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 Ni-NTA spin column] with 600 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-20_.28wash_buffer.29 NPI-20] and centrifuge for 2 min at 890 g (4 °C). Repeat this step and collect flow-through each time.
 
-
**Elute the protein with 300 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-500_.28elution_buffer.29 NPI-500] and centrifuge for 2 min at 890 g (4 °C). Repeat this step and collect flow-through each time.
 
-
** For centrifugal ultrafiltration use [http://www.sartorius-stedim.com/DE/en/Centrifugal-Ultrafiltration--Vivaspin-%26-Centrisart/Vivaspin-500/VS0111-VIVASPIN-500-5%2C000-MCWO-PES-25-BOX/6htcki6uex7/m8qc5rabaox/n8uj9p1cyz4/article.htm Vivaspin 500] concentrators ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) with a 5000 molecular weight cut-off PES membrane. Repeat the procedure three times by adding [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#DNA_ligase_buffer DNA ligase buffer] (4 °C) for buffer exchange (remove of imidazole).
 
-
** Check each purification step and especially the purity of the protein in the final condition by [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE analysis].
 
-
** Determine the protein concentration with a [https://2011.igem.org/Team:Bielefeld-Germany/ Bradford Protein Assay].
 
-
** For long-time storage keep the protein at -20 °C.
 
-
** If the binding conditions are not effective enough you can reduce the imidazole concentration in [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10] to 1-5 mM. If the eluate contains unspecifically bound proteins maybe a washing step with [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10] containing 50 mM or 100 mM imidazole could help to get higher purity.
 
-
*Large-scale purification
+
===Immobilization of SgsE on silica beads===
-
**Resuspend a cell culture pellet in 5 ml [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer] per gramm biomass.
+
* after purification: dialyse 18 h at 4 °C against ddH<sub>2</sub>O in the dark
-
**Add 10 mM MgCl<sub>2</sub>, 1 mg/ml lysozyme] as well as 5 Units benzonase nuclease per 1 ml cell culture volume and mix thoroughly.
+
* centrifuge and use supernatant -> this is the monomeric protein solution
-
**Incubate at 4 °C for 30 min.
+
* measure protein concentration
-
**Centrifuge the lysate for 30 min at 10000 g (4 °C). Collect the supernatant (cleared lysate).
+
* dilute purified monomeric S-layer solution to 1 mg mL<sup>-1</sup> protein with ddH<sub>2</sub>O -> store in the dark at 4 °C
-
**Prepare a [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&moduleid=165904 HisTrap™ FF crude column] ([http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/life-sciences_homepage GE Healthcare]) by equilibration with 5 column volumes of [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer]. The following steps have to be performed at 4 °C.
+
* suspend silicium dioxide beads in [[Team:Bielefeld-Germany/Protocols/Materials#Hanks_Buffered_Saline_Solution_.28HBSS.29 | HBSS]] (pH 7.4) and mix it with the 1 mg mL<sup>-1</sup> S-layer solution
-
**Load the cleared lysate onto the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 HisTrap™ FF crude column] and collect flow-through.
+
** ratio of beads to protein can be varied
-
**Wash the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 HisTrap™ FF crude column] with 10 column volumes of [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer] and collect flow-through.
+
** 0.1 mg mL<sup>-1</sup> final protein concentration
-
**Elute the protein successively with 5 ml of buffers that contain different concentrations of imidazole (e.g. [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer] with 40, 60, 100, 300 and 500 mM imidazole) and collect flow-through in 1 ml fractions.
+
** contact with [[Team:Bielefeld-Germany/Protocols/Materials#Hanks_Buffered_Saline_Solution_.28HBSS.29 | HBSS]] buffer will start assembly of SgsE
-
** For centrifugal ultrafiltration use [http://www.sartorius-stedim.com/DE/de/Zentrifugal-Ultrafiltration--Vivaspin-%26-Centrisart/Vivaspin-15/VS1501-VIVASPIN-15-10%2C000-MWCO-PES-10-BOX/6htcki6uex7/1b11mt9gskl/kn4oouwu6g3/article.htm Vivaspin 15] concentrators ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) with a 10000 molecular weight cut-off PES membrane. Repeat the procedure three times by adding [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#DNA_ligase_buffer DNA ligase buffer] (4 °C) for buffer exchange (remove of imidazole).
+
* incubate on vertical rotator at room temperature for 4 h
-
** Check each purification step and especially the purity of the protein in the final condition by [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE analysis].
+
* after incubation: centrifuge down the beads (1 min, > 15000 g), wash them twice with ddH<sub>2</sub>O and store them afterwards in ddH<sub>2</sub>O at 4 °C in the dark
-
** Determine the protein concentration with a [https://2011.igem.org/Team:Bielefeld-Germany/ Bradford Protein Assay].
+
-
** For long-time storage keep the protein at -20 °C.
+
-
===Deadenylation===
 
-
The majority of purified DNA ligase from [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX] is usually in the adenylated form. But only the apoenzyme is really useful for the [http://pubs.acs.org/doi/abs/10.1021/ac102742k NAD<sup>+</sup> bioassay] so that the AMP moiety has to be removed.
+
===Immobilization of SbpA on silica beads===
 +
* after purification: dialyse 18 h at 4 °C against ddH<sub>2</sub>O in the dark
 +
* centrifuge and take supernatant -> this is the monomeric protein solution
 +
* measure protein concentration
 +
* dilute purified monomeric S-layer solution to 1 mg mL<sup>-1</sup> protein with ddH<sub>2</sub>O -> store in the dark at 4 °C
 +
* suspend silicium dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl<sub>2</sub>) and mix it with the 1 mg mL<sup>-1</sup> S-layer solution
 +
** ratio of beads to protein can be varied
 +
** 0.1 mg mL<sup>-1</sup> final protein concentration
 +
** contact with recrystallization buffer will start assembly of SbpA
 +
* incubate on vertical rotator at room temperature for 4 h
 +
* after incubation: centrifuge down the beads (1 min, > 15,000 g), wash them twice with ddH<sub>2</sub>O and store them afterwards in ddH<sub>2</sub>O at 4 °C in the dark
-
** Mix the purified DNA ligase with [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Deadenylation_buffer deadenylation buffer] and incubate for 10 min at 37 °C.
 
-
** Use [http://www.sartorius-stedim.com/DE/de/Zentrifugal-Ultrafiltration--Vivaspin-%26-Centrisart/Vivaspin-15/VS1501-VIVASPIN-15-10%2C000-MWCO-PES-10-BOX/6htcki6uex7/1b11mt9gskl/kn4oouwu6g3/article.htm Vivaspin 15] concentrators ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) with a 10000 molecular weight cut-off PES membrane for buffer exchange at 4 °C. Repeat the procedure at least three times by adding [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#DNA_ligase_buffer DNA ligase buffer] (remove of NMN and formed NAD<sup>+</sup>).
 
-
** Check the deadenylation process by [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE analysis]. If the deadenylation was successful there should not be a double band indicating both forms of DNA ligase.
 
-
** Determine the protein concentration with a [https://2011.igem.org/Team:Bielefeld-Germany/ Bradford Protein Assay].
 
-
** For long-time storage keep the protein at -20 °C.
 
-
===NAD<sup>+</sup> bioassay===
+
===Immobilization of SgsE on silicon wavers===
 +
* used wavers: p-type / bor
 +
* wash wavers with 70 % ethanol and ddH<sub>2</sub>O
 +
* cover the waver with 0.1 mg mL<sup>-1</sup> purified protein in [[Team:Bielefeld-Germany/Protocols/Materials#Hanks_Buffered_Saline_Solution_.28HBSS.29 | HBSS]] buffer for 4 h
-
** Prepare a 80 μl aliquot composed of 250 nM molecular beacon and 250 nM split target in [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer].
 
-
** Incubate for 8 min at 37 °C.
 
-
** Add 6 Units of deadenylated DNA ligase (approximately ... ng).
 
-
** Monitor the fluorescence with a spectrofluorometer at 37 °C and wait until it reaches equilibrium.
 
-
** Add NAD<sup>+</sup> in different final concentrations (e.g. 0, 0.3, 0.5, 5, 10, 30, 40, 80, 200, 300, 500, 1000 nM), stirr shortly and monitor the fluorescence at 37 °C again.
 
-
** Determine the initial enhancement rate of flourescence intensity for each NAD<sup>+</sup> concentration.
 
-
** For the calibration curve plot the initial velocity against the NAD<sup>+</sup> concentration.
 
-
<br style="clear: both" />
+
===Immobilization of SbpA on silicon wavers===
 +
* used wavers: p-type / bor
 +
* wash wavers with 70 % ethanol and ddH<sub>2</sub>O
 +
* cover the waver with 0.1 mg mL<sup>-1</sup> purified protein in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl<sub>2</sub>) for 4 h

Latest revision as of 01:53, 29 October 2011

Production Protocols: These are the protocols for the cultivation and the downstream processing.

Contents

Cultivation

Expression of S-layer genes in E. coli

  • Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]
  • Medium: LB medium supplemented with 20 mg L-1 chloramphenicol or autoinduction medium
    • Cultivations in LB-medium were supplemented with 0.1 % L-rhamnose as inducer, when the designated OD600 was reached.
    • Autoinduction medium for expressing <partinfo>K525304</partinfo>, <partinfo>K525305</partinfo>, <partinfo>K525306</partinfo>, <partinfo>K525405</partinfo>, was supplemented with 1 mM IPTG.

For characterising the expression rate and the influance on E. coli growth behavior an automatic sampling system (Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath BU) was used. These cultivations were carried out in an Infors AG AQUATRON.

  • 150 mL culture in 500 mL shaking flask with baffles (Schott) with silicon plugs
  • Cultivation temperature: 37 °C at 120 rpm


Expression of bisphenol A degrading BioBricks in E. coli


  • Used BioBricks: , <partinfo>K525512</partinfo>, <partinfo>K525517</partinfo>, <partinfo>K525552</partinfo>
  • Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]
  • Medium: LB medium supplemented with 100 mg L-1 ampicillin and 120 mg L-1 bisphenol A
    • BPA is thermally stable -> you can autoclave it together with the medium
  • 100 mL culture in 300 mL shaking flask without baffles (Schott) with silicon plugs
  • Cultivation temperature: 24 °C, 30 °C or 37 °C, tempered with Infors AG AQUATRON at 120 rpm
  • for characterizations: automatic sampling every three hours with Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath BURMA-SHAVE!!
    • the characterization experiment setup is shown on the picture on the right


Bioreactor cultivations with E. coli KRX

To obtain higher amounts and concentration of proteins we cultivated and expressed in a bioreactor. It is possible to cultivate several liters and to control temperature, pH and DO.

  • Bioreactor: Bioengineering NLF22 7 L or [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with Bioengineering DCU
  • Medium: HSG medium with 20 mg L-1 chloramphenicol or 100 mg L-1 ampicillin
  • Culture volume: 2.5 - 5 L
  • Starting OD600: 0.1 - 0.4
  • DO: 40 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
  • pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
  • Antifoam: BASF pluronic PE-8100
  • Induction after 4 h cultivation time with 0.2 % rhamnose and 1 mM IPTG (in culture medium)
  • Harvest after 8 - 13 h


Purification methods

Enzymatic cell lysis with lysozyme

  • After cultivation biomass was collected by centrifugation at 5,000 g at 4 °C for 20 min.
  • 1 g of biomass (wet weight) was suspended in 10 mL of enzyme buffer containing 0.1 % Triton X-100, 2 µL benzonase (250 U/µL) and 40 µL of lysozyme (100 mg mL-1)
  • Incubation for 30 min at 4 °C
  • reaction mixture was centrifuged for 30 - 90 min at 15,000 g at 4 °C


Release of periplasmic protein fraction from E. coli by cold osmotic shock

Modified protocol from [http://www.jbc.org/content/240/9/3685.full.pdf+html?sid=4a90c176-0ec3-489f-8c82-4734274cebf5 Neu & Heppel, 1965].

  • Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells.
  • Discard the entire supernatant.
  • Resuspend the cells ice-cold cell fractionating buffer 1. The resulting volume should be 1/4 of the former suspension volume.
  • Incubate for 20 min on ice. Ivert the suspension at regular intervals to counteract sedimentation.
  • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C).
  • Discard the entire supernatant.
  • Resuspend the cells ice-cold cell fractionating buffer 2. The resulting volume should be 1/4 of the former suspension volume.
  • Incubate for 10 min on ice under regular invertion.
  • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C).
  • Save the supernatant, which contains the periplasmatic proteins.
  • If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter.


Inclusion body clean-up

  • harvest the cells by centrifugation (30 min, 10,000 g, 4 °C)
  • resuspend pellet and disrupt cells
  • centrifuge lysate (60 - 90 min, >17000 g, 4 °C)
  • wash pellet at least two times with water to remove water-soluble proteins
  • after washing the pellet: incubate the pellet in denaturation buffer for 60 min, 4 °C with vertical rotator
    • final concentration in denaturation buffer: 0.5 mg wet biomass per mL
  • centrifuge (60 min, >17,000 g, 4 °C)
    • the higher the speed, the better the result
  • collect supernatant and incubate the pellet again in denaturation buffer (60 min, 4 °C, vertical rotator)
  • centrifuge (60 min, >17,000 g, 4 °C)
  • collect supernatant and discard pellet


Ammonium sulfate precipitation

  • Mix fraction you want to clean-up with ammonium sulfate
    • To precipitate S-layer proteins from Corynebacterium, 40 % ammonium sulfate saturation concentration is a good concentration (247 g L-1 ammonium sulfate at 25 °C)
  • Incubate 30 min at room temperature on a shaker
  • Centrifuge (the faster and longer the better) and solve the precipitate in water or buffer


Ultra-/Diafiltration


  • Arrange the filtration module as shown on the right side.
  • Microfiltration (0.22 µm) or cross flow filtration with 300 kDa (we used a Milipore Pellicon XL 300) membrane of sample before ultrafiltration.
  • For concentrating the sample just filter it until the desired volume is left in the feed reservoir. For diafiltration (e.g. buffer exchange, desalting) dilute the feed reservoir several times and filter continously.
  • Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
    • 50 or 100 kDa cut-off
    • 50 cm2 filtration area
    • tangential flow filter
    • Hydrophilic polyvinylidene fluoride membrane
  • Used pump: SciLog TANDEM 1081 peristaltic pump
    • flow rate during filtration: 40 mL min-1


Ion exchange chromatography (IEX) for S-layer proteins from Corynebacterium

  • used column: DEAE HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
  • flow rate: 1 mL min-1
  • equilibrate column with > 10 column volumes of binding buffer
  • inject sample and wash column with binding buffer until UV signal is constant
  • elute with 20 %, 40 % and 60 % of binding / elution buffer mix and collect fractions
  • elute remaining proteins with 100 % elution buffer


Ion exchange chromatography (IEX) for S-layer proteins from Lysinibacillus sphaericus

  • tested with <partinfo>K525405</partinfo>
  • used column: DEAE HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
  • flow rate: 0.5 mL min-1
  • equilibrate column with 20 column volumes of binding buffer
  • inject sample and wash column with binding buffer until UV signal is constant
  • elute with 10 % of binding / elution buffer mix and collect fraction
  • elute remaining proteins with 100 % elution buffer


Hydrophobic Interaction Chromatography (HIC) for S-layer proteins

  • tested with <partinfo>K525405</partinfo> and <partinfo>K525311</partinfo>
  • used column: Butyl HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
  • flow rate: 0.5 mL min-1
  • equilibrate column with 20 column volumes of binding buffer
  • inject sample and wash column with binding buffer
  • elute in 10 % steps of binding / elution buffer mix and collect fractions
    • <partinfo>K525405</partinfo> elutes at 70 % buffer B
    • <partinfo>K525311</partinfo> elutes at 50 % buffer B
  • elute remaining proteins with 100 % elution buffer


His-tag affinity chromatography

  • Column: 1 mL HisTrap FF crude by [http://www.gehealthcare.com/ GE Healthcare]
  • Harvest cells by centrifugation at 10,000 g for 10 min at 4 °C
  • Discard the supernatant and freeze bacterial pellet at -20 °C for at least 30 min
  • Resuspend the pellet in 5 mL binding buffer for each gram of cell paste
  • Wash column with 5 - 10 mL of deionized water
  • Equilibrate column with 5 - 10 mL of binding buffer

denaturing

  • For buffers see table buffers for his-tag affinity chromatography
  • Mechanical lysis:
    • Sonification on ice for 6 - 10 min with Sonifier 450 by [http://www.gehealthcare.com/ Branson], max. 20 W, cooled on ice
  • Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
  • Centrifuge at 15,000 g for 30 min at 4 °C
  • Filter sample (sterile filter or 300 kDa cut-off)


  • Syringe method
    • Equilibrate with binding buffer
    • Load sample onto column
    • Wash with 10 mL binding buffer
    • Elute with 5 mL of elution buffer with increasing imidazole concentrations
    • Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction
    • Re-equilibrate the column with binding buffer


  • ÄKTA method
    • Equilibrate with 20 column volumes binding buffer, 0.5 mL min-1
    • Load sample onto column
    • Wash with binding buffer until UV signal is constant
    • Elute with 50 mM imidazol
    • Elute remaining proteins with 500 mM imidazol


non-denaturing

  • For buffers see table buffers for his-tag affinity chromatography
  • Enzymatic lysis:
    • Add 0.2 mg L-1 lysozyme, 3 units of Benzonase per mL of culture volume and 1 mM MgCl2
    • Stirr for 30 in at 4 °C
  • Centrifuge at 10,000 g for 30 min at 4 °C
  • Load sample onto the column
  • Wash with 10 mL binding buffer
  • Elute with 5 mL of elution buffer with increasing imidazole concentrations
  • Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction
  • Re-equilibrate the column with binding buffer


Production and purification strategies

Fusion proteins of SgsE and SbpA

  • Cultivation
    • Bioreactor: Bioengineering NLF22 7 L or [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] with Bioengineering DCU
    • Medium: HSG medium with 20 mg L-1 chloramphenicol or 100 mg L-1
    • Culture volume: 2.5 - 5 L
    • Inoculation OD600: 0.1 - 0.4
  • DO: 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
  • pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
  • Antifoam: BASF pluronic PE-8100
  • Induction after 4 h cultivation time with 0.2 % rhamnose and 1 mM IPTG (in culture medium)
  • Harvest after 8 - 13 h
  • Cell lysis
    • Centrifuge down the cells (10,000 g, 30 min, 4 °C)
    • Resuspend pellet in enzyme buffer or binding buffer for denaturing his-tag purification
    • Cell lysis with high-pressure homogenizer (800 bar, 3 cycles at 4 °C)
    • Centrifuge down the lysate (10,000 g, 60 min, 4 °C)
  • Inclusion body clean-up
    • wash pellet from cell lysis with water twice
    • after washing the pellet: incubate the pellet in denaturation buffer for 60 min, 4 °C with vertical rotator
      • final concentration in denaturation buffer: 0.5 mg wet biomass per mL
    • centrifuge (60 min, >17,000 g, 4 °C)
      • in general with all centrifugations during this clean-up: the higher the speed, the better the result
    • collect supernatant and incubate the pellet again in denaturation buffer (60 min, 4 °C, vertical rotator)
    • centrifuge (60 min, >17,000 g, 4 °C)
    • collect supernatant and discard pellet


  • Filtration
    • Arrange the filtration module as shown on the right side.
    • Collect permeate of cross flow filtration with 300 kDa membrane of sample before ultrafiltration
      • This step is for removing cell debris
    • Diafiltrate with 100 kDa membrane against denaturation buffer
      • constantly delute permeate with the buffer, keeping the permeate volume as low as possible
    • Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
      • 50, 100 or 300 kDa cut-off
      • 50 cm2 filtration area
      • tangential flow filter
      • Hydrophilic polyvinylidene fluoride membrane
    • Used pump: SciLog TANDEM 1081 peristaltic pump
      • flow rate during filtration: 40 mL min-1

OR WHEN USING A HIS-TAGGED PROTEIN (RECOMMENDED):

  • His-6-affinity tag purification
    • For buffers see table buffers for his-tag affinity chromatography
    • Mechanical lysis as described above in binding buffer
    • Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
    • Centrifuge at 15,000 g for 30 min at 4 °C
    • Filter sample (sterile filter or 300 kDa cut-off)
    • Equilibrate column (1 mL HisTrap FF crude from GE Healthcare) with 20 column volumes binding buffer, 0.5 mL min-1
    • Load sample onto column
    • Wash with binding buffer until UV signal is constant
    • Elute with 50 mM imidazol
    • Elute remaining proteins with 500 mM imidazol
  • Dialysis
    • Fill retentate from DF/UF or chromatography elution fraction in dialysis tube ([http://www.carl-roth.de/ Roth], cellulose, 10 kDa cut-off)
    • Dialyse against ddH2O for 18 h at 4 °C in the dark
    • After dialysis: centrifuge down the precipitation (45 min, 17,000 g, 4 °C) and collect the supernatant
    • Measure protein concentration in supernatant, dilute to 1 mg mL-1 with ddH2O and store at 4 °C in the dark
  • Scheme of purification strategy for S-layer (fusion) proteins:

With inclusion body purification:

Bielefeld-Germany2011-305 405-Aufreinigung symbol.png

With HisTrap:

IGEM-Bielefeld2011-322 Aufreinigung symbol.png
  • Further purification of dirty inclusion body purification by IEX and HIC as described above


Fusion proteins of CspB without lipid anchor with TAT-sequence

  • Cultivation
    • 150 mL culture in 500 mL or 300 mL culture in 1000 mL shaking flasks with baffles (Schott) with silicon plugs
    • Medium: autoinduction medium
    • Cultivation temperature: 37 °C at 120 rpm
  • Supernatant precipitation
    • Centrifuge down the cells (10000 g, 30 min, 4 °C) and collect the supernatant
    • To precipitate S-layer proteins from Corynebacterium, i.e. 40 % ammonium sulfate saturation concentration (247 g L-1 ammonium sulfate at 25 °C)
    • Incubate 30 min at room temperature on a shaker
    • Centrifuge (the faster and longer the better) and solve the precipitate in binding buffer for IEX


  • Filtration
    • Arrange the filtration module as shown on the right side.
    • Collect permeate of cross flow filtration with 300 kDa membrane of sample before ultrafiltration
      • This step is for removing cell debris
    • Diafiltrate with 50 kDa membrane against binding buffer for IEX
      • constantly delute permeate with the buffer, keeping the permeate volume as low as possible
    • Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50], XL 100 or XL 300 membranes
      • 50, 100 or 300 kDa cut-off
      • 50 cm2 filtration area
      • tangential flow filter
      • Hydrophilic polyvinylidene fluoride membrane
    • Used pump: SciLog TANDEM 1081 peristaltic pump
      • flow rate during filtration: 40 mL min-1
  • Ion exchange chromatography
    • used column: DEAE HiTrap 1 mL with [http://www.gehealthcare.com/ GE Healthcare] ÄKTA
    • flow rate: 1 mL min-1
    • equilibrate column with 10 column volumes of [Team:Bielefeld-Germany/Protocols#Buffers_for_S-layer_IEX | binding buffer]
    • inject sample and wash column with binding buffer
    • elute with 20 %, 40 % and 60 % binding / elution buffer mix and collect samples -> take the cleanest fraction for further work
    • elute remaining proteins with 100 % elution buffer
  • Scheme of purification strategy for CspB S-layer (fusion) proteins:
Bielefeld-Germany2011-CH4-Aufreinigung symbol.png


Recrystallization of S-layer proteins

Recrystallization of SgsE and SbpA in solution

  • after purification: dialyse 18 h at 4 °C against ddH2O in the dark
  • centrifuge and use supernatant -> this is the monomeric protein solution
  • dialyse 18 h at 4 °C against
    • HBSS pH 7.4 for SgsE
    • recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl2) for SbpA


Immobilization of SgsE on silica beads

  • after purification: dialyse 18 h at 4 °C against ddH2O in the dark
  • centrifuge and use supernatant -> this is the monomeric protein solution
  • measure protein concentration
  • dilute purified monomeric S-layer solution to 1 mg mL-1 protein with ddH2O -> store in the dark at 4 °C
  • suspend silicium dioxide beads in HBSS (pH 7.4) and mix it with the 1 mg mL-1 S-layer solution
    • ratio of beads to protein can be varied
    • 0.1 mg mL-1 final protein concentration
    • contact with HBSS buffer will start assembly of SgsE
  • incubate on vertical rotator at room temperature for 4 h
  • after incubation: centrifuge down the beads (1 min, > 15000 g), wash them twice with ddH2O and store them afterwards in ddH2O at 4 °C in the dark


Immobilization of SbpA on silica beads

  • after purification: dialyse 18 h at 4 °C against ddH2O in the dark
  • centrifuge and take supernatant -> this is the monomeric protein solution
  • measure protein concentration
  • dilute purified monomeric S-layer solution to 1 mg mL-1 protein with ddH2O -> store in the dark at 4 °C
  • suspend silicium dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl2) and mix it with the 1 mg mL-1 S-layer solution
    • ratio of beads to protein can be varied
    • 0.1 mg mL-1 final protein concentration
    • contact with recrystallization buffer will start assembly of SbpA
  • incubate on vertical rotator at room temperature for 4 h
  • after incubation: centrifuge down the beads (1 min, > 15,000 g), wash them twice with ddH2O and store them afterwards in ddH2O at 4 °C in the dark


Immobilization of SgsE on silicon wavers

  • used wavers: p-type / bor
  • wash wavers with 70 % ethanol and ddH2O
  • cover the waver with 0.1 mg mL-1 purified protein in HBSS buffer for 4 h


Immobilization of SbpA on silicon wavers

  • used wavers: p-type / bor
  • wash wavers with 70 % ethanol and ddH2O
  • cover the waver with 0.1 mg mL-1 purified protein in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl2) for 4 h