Team:Freiburg/Labjournals/ProtPur/Before July
Protein Purification
Protein purification LabJournal entries before July 2015
30.06.2015
pIG15_1301 C43
in this experiment the protein anti dihydroxyacid dehydratase (His-Tag) should be purified by Ni-NTA
- 500ml culture was harvested by centrifugation, 5000xg, 20min, 4°C
- for cell disruption the pellet (4g) was resuspended in 30ml lysis buffer (20mM NaH2PO4, 300mM NaCl, 10mM Imidazol, pH 8,0)
- sonification was performed 5min, 6xcycles on ice
- afterwards 1:1000 PMSF (stock 300mM) was added
- to get rid of the inclusion bodies, the cell lysate was centrifuged 10 000xg, 30min, 4°C
- the supernatant was used for the protein purification
Protein purification
- 400ul slurry of the Ni-NTA beads were equilibrated with 2ml lysis buffer (2x times)
- supernatant and beads were incubated for 1h, 4°C under rotating conditions
- by centrifugation (500xg, 1min, 4°C) the flowthrough was taken und stored on ice for furhter SDS-PAGE Analysis
- the beads were washed five times with wash buffer (20mM NaH2PO4, 300mM NaCl, 20mM Imidazol, pH 8,0)
- to improve the washing step the imidazole concentration was increased up to 30mM for one washing step
- for the elution of anti dehydroxyacid dehydratase, 500ul of the elution buffer (20mM NaH2PO4, 300mM NaCl, 500mM Imidazol, pH 8,0) was used. The elution was performed five times.
- the samples are stored at -80°C
Results
To confirm, whether the gIII (secretion signal to periplasm) is cleaved off, western blot should be performed with anti-His Tag antibdoy.
pET_Spy catcher C43
- transformation of the pET_Spy catcher in C43 competent cells (Amp+Cml)
29.06.2015
pIG15_1301 C43
- 500ml LB-medium was inoculated with 5ml o/n culture C43+pIG15_1301 [Amp+Cml]
- cells were grown until OD600~0.5
- the expression of the protein was induced with 0.5mM IPTG and set to 30°C o/n
pQE GFP nT, pQE GFP H2x6His, pQE HA mCherry 2x6His
- cells were harvested by centrifugation (20 min, 5000 g)
- supernatant was slightly colored but discarded
- cells were resuspended in 10 mL lysis buffer per 100 mL cell culture
- disruption with sonification (70% power, 5 min, mode 6)
- removal of cell debris by centrifugation (4°C, 15000 g, 30 min)
- supernatant was aliquoted (1 mL) and shock frozen in liquid nitrogen
- stored @ -20 °C
- glycerol stocks of all three transformations were done (shock freeze in liquid nitrogen, store @ -80°C)
28.06.2015
pIG15_1301 C43
glycerol stock was done (shock freeze in liquid nitrogen, store @ -80°C)
Experiment
- 30°C sample (25ml) was harvested by centrifugation, 20 min 5000xg, separated in 10 ml fractions
- the pellets of 10 ml cell culture were treated differently → cell disruption by sonification and periplasmic preparation, respectively.
- for sonification the pellets of 30°C and 37°C were resuspend in 5ml lysis puffer (20mM NaH2PO4, 300mM NaCl, 10mM Imidazol)
- sonification: 5 min, 6xcycle
- addition of PMSF (stock 300mM) 1:1000
- centrifugation 10 000xg, 30min, 4°C to pellet the inclusion bodies
- the supernatant was transfered into a new falcon, pellet was resuspended in the equal volume of lysis buffer
- periplasmic preparation was performed accroding to the protocol. periplasmic preparation
- 12.5% SDS-PAGE Analysis was realized to check, whether the protein is soluble as well for comparison of the different cell disruption methods
Result
- anti-dehydroxyacid dehydratase is about 74kDa.
- the protein seems to soluble.
- some mistakes occur during the loaoding process of the samples
pIG15_1301 C43
- 5ml o/n culture of C43 + pIG15_1301 for large scale over expression [Amp+Cml]
pQE GFP nT, pQE GFP H2x6His, pQE HA mCherry 2x6His
- 1 Ml of o/N cultures was used to inoculate 100 mL LB-Medium (amp) for day-culture
- colonies were grown at 37°C 200 rpm
- at an OD600 of 0.5 (228 min post inoculation) samples were induced with 1 mM IPTG
- culture growth was followed by OD-measurement
- o/N cultures were set up for glycerol stocks
27.06.2015
pIG15_1301 C43
- 2x 25 ml LB-Medium inoculated with 1ml o/n culure, grown until OD600 ~ 0.5, 37°C
- induced with IPTG 0.5mM and 1mM, respectively.
- samples were splitted to two different temperatures: 37°C (0.5 mM IPTG) + 30°C (1mM IPTG)
- sample with the condition 37°C + 0.5mM IPTG were harvested 4h after induction
- the 30°C, 1mM IPTG sample was grown over night.
Competent cells TOP10 + C43
- competent cells were performed according to the protocol competent cells
pQE GFP nT, pQE GFP H2x6His, pQE HA mCherry 2x6His
- two colonies from each transformation were picked an striven out on plate
- plates were grown over day
- from each plate (and thus from each plasmid) one 3mL o/N culture was set up
26.06.2015
- o/n culture of C43 (Cml resistance) for competent cells [5ml]
- o/n culture of TOP10 cells for competent cells [5ml]
- o/n culture C43 (Cml) + pIG15_1301 (antibody for dihdroxyacid dehydratase) (Amp) [2ml]
pQE GFP nT, pQE GFP H2x6His, pQE HA mCherry 2x6His
- plasmids were obtained from AG Roth
- Transformation with Rosetta cells
- grown o/n on plate
25.06.2015
- TOP10 + C43 (new expression strain from Toolbox) on plate with no and Cml, respectively.
pIG15_1301 C43
In this experiment VL and VH [anti dehydroxy dehydratase] (pHAL15 plasmid received from the research group Meyer et al.) should be expressed in E.coli. The protein itself (pIG15_1501) is purified already and stored in -80°C; but not desalted!
- Trafo acorrding to the protocoll C43 (Cml) + pIG15_1301 (antibody for dihdroxyacid dehydratase) (Amp)
- C43 with pRARE plasmid: new expression strain (Toolbox)
- recommendation: two conditions:
- 1. after induction (1mM IPTG) 30°C, o/n
- 2. after induction (0.5mM IPTG) 37°C, 4h
10.06.2015
pIG15_1601 BL21: cell lysis
Experiment
- Cell lysis was performed according to cell disruption protocoll
- SDS-PAGE on 8.5 % Gel was performed according to SDS-PAGE protocol
Results
Also after changing the expression strain to BL21 there seems to be no effective expression of the protein in the soluble fraction.
09.06.2015
pIG15_1601 BL21: culture
Experiment
- 4x 100 mL LB-medium 100 µg/mL Amp & 25 µg / mL Cml inoculated with 1 mL o/n culture per flask
- growth of culture until OD600 = 0.5
- Induction with 0.1 mM or 0.01 mM IPTG for half of the flasks each
- took samples (10 mL) before induction, after 2h & 4h after induction
- harvested cells were stored on ice
08.06.15
Purification pIG15_1601 Rosetta
Experiment
- 2x 500 µL regenerated Ni-NTA beads were equilibrated in Lysis buffer by 3x washing with Lysis buffer
- Cell lysate supernatants from the 2h and 4h samples of 18°C or 24°C respectively were combined and inoculated with ~ 500 µL of regenerated bead suspension
- samples were incubated o/n on 4°C with slight agitation
Rock'N'Roller stood still the next morning
- purification was performed as described in bead_purification
- beads were regenerated as described in the Ni-NTA bead regeneration protocol
Results
Resulting protein concentration: blanked with elution buffer
| Temperature | Fraction | c [mg/mL] | 260/280 |
|---|---|---|---|
| 18°C | E1 | 0.049 | 1.27 |
| 18°C | E2 | 0.032 | 1.36 |
| 18°C | E3 | 0.036 | 1.65 |
| 18°C | E4 | 0.010 | 2.39 |
| 18°C | E5 | 0.004 | 4.59 |
| 28°C | E1 | -0.059 | 0.15 |
| 28°C | E2 | -0.013 | -2.02 |
| 28°C | E3 | -0.004 | -6.07 |
| 28°C | E4 | -0.008 | -2.05 |
| 28°C | E5 | -0.002 | -9.54 |
A control with RNAse free water at the beginning, in the middle and at the end of the measurement resultet in consistent values of about -0.058 mg/L. A measurement of the elution buffer at the end resulted in -0.01.
→ The measurement itself seems reliable
After Elution with 500 mM imidazole there is protein visible at 65 kDa, the actual weight of the pIG15_1501 Salmonella-Antigen.
Still no reliable amount of enriched protein visible!
Protein visible at the weight of the pIG15_1501 antigen → beads were not clean after regeneration
Next time: include an additional debinding step using a very high imidazole concentration before regeneration of the beads
Protein visible at the weight of the pIG15_1501 antigen → beads were not clean after regeneration
Next time: include an additional debinding step using a very high imidazole concentration before regeneration of the beads
pIG15_1601 BL21 culture
Experiment
- started o/n culture of pIG15_1601 BL21 pLys in AMp/Cml
05.06.15
pIG15_1601 Rosetta
considerations
- To check for the induction of the transgenic sequence a whole cell lysate was performed
- The localization of the protein in inclusion bodies should be checked on by differential centrifugation
Results
In the whole cell lysates there is no clear protein band visible
Thought of a double band wherefrom the upper on may be consist of the desired protein of about 85 kDa (signified with arrows)
→ Try the purification of the supernatants from the overepxression test for further confirmation
Thought of a double band wherefrom the upper on may be consist of the desired protein of about 85 kDa (signified with arrows)
→ Try the purification of the supernatants from the overepxression test for further confirmation
04.06.15
pIG15_1601 Rosetta
Experiment
- 12% SDS-PAGE Analysis for 1 ml samples of pIG15_1601 from 3.6 was performed (15µl sample, 4µl marker was applied)
- frozen and o/n pellets were resuspend in 5 ml lysis buffer and sonificated
- PMSF (300mM Stock) 1:1000 was added
- 12% SDS-PAGE Analysis was performed
03.06.15
pIG15_1601 Rosetta
Experiment
- 5x 100 ml LB-medium was inoculated with 1ml o/n culture and grown at 37°C until the OD600 ~ 0.5
- cells were induced with 1mM and 0.1mM IPTG, respectively
- and distributed to different temperatures: 37°C, 28°C and 18°C each with one IPTG concentration, for 37°C only 0.1 mM IPTG was used
- take 10 ml and 1 ml samples at each timepoint (2h, 4h and o/n after induction)
- centrifuge the 10ml samples for 20min at max. speed
- the 1ml samples of each timepoint for SDS-PAGE Analysis, → centrifuge at maximum speed, discard supernatant and add 200 of 2,5x SDS Loading Dye. Boil samples for 10 min, 95°C
01.06.15
pIG15_1601 Rosetta
Experiment
- streaked pIG15_1601 in Rosetta (Glycerolstock) on Cml/Amp plate → for further overespression experiments (testing several temperatures)
30.05.15
- SDS-PAGE with samples from 29.05.
- Coomassie-Staining for 20 min
- Destaining
29.05.15
bead regeneration
Ni-NTA-beads were regenerated according to bead regeneration protocol.
pIG15_1501 and pIG15_1601: protein purification
Experiment
- the o/n incubated beads were centrifuged for 1min, 500xg; the supernatant was labeld as flow-through and stored on ice
- 2ml of wash buffer (20mM NaH2PO4, 300mM NaCl, 20mM IPTG) was added, 1min, 500xg
- the supernatant was taken off and collected for further analysis
- the washing step was repeated 5x times
- for elution the buffer of 20mM NaH2PO4, 300mM NaCl and 500mM Imidazole was used.
- elution was performed in 500ul in five steps
- for analysis of the purification a SDS-PAGE Gel (12%) was used
- of each step 9ul sample + 6ul 2,5x SDS-Loading Dye was used
Results
protein concentration in eluate pIG15_1501
| Eluate # | Concentration [mg/mL] | 260/280 |
|---|---|---|
| 1 | 10 | 0.62 |
| 2 | 7.39 | 0.75 |
| 3 | 2.59 | 0.71 |
| 4 | 0.91 | 0.70 |
| 5 | 0.45 | 0.78 |
Using NanoDrop 2000c
protein concentration in eluate pIG15_1601
| Eluate # | Concentration [mg/mL] | 260/280 |
|---|---|---|
| 1 | 0.34 | 1.89 |
| 2 | 0.22 | 1.83 |
| 3 | 0.52 | 1.80 |
| 4 | 0.31 | 1.89 |
| 5 | 0.20 | 1.98 |
Using NanoDrop 2000c
The 63.81 kDa His-tagged putative dihydroxyacid dehydratase (S. typhimurium) was clearly enriched in the gel what could be confirmed in nanodrop-analysis!
For pIG15_1601 there is no enrichtment visible
The unspecific binding of E. coli proteins in this sample may be used to estimate unspecific signal
For pIG15_1601 there is no enrichtment visible
The unspecific binding of E. coli proteins in this sample may be used to estimate unspecific signal
pIG15_803: Cell lysis
Experiment
deviation from cell lysis protocol:
- frozen pellets were resuspend in 5 ml lysis buffer and sonificated
- PMSF (300mM Stock) 1:1000 was added
- 12% SDS-PAGE Analysis was performed
Results
There is no enrichment visible of the 15 kDa target protein
28.05.15
pIG15_803: overexpression test
Experiment
- 6x 100 ml LB-medium was inoculated with 1ml o/n culture and grown at 37°C until the OD600 ~ 0.5
- cells were induced with 1mM and 0.1mM IPTG, respectively
- and distributed to different temperatures: 37°C, 28°C and 18°C each with one IPTG concentration
- take 10 ml samples at each timepoint (2h, 4h and o/n after induction)
- centrifuge the 10ml samples for 20min at max. speed
- the 1ml samples of each timepoint for SDS-PAGE Analysis, → centrifuge at maximum speed, discard supernatant and add 200 of 2,5x SDS Loading Dye. Boil samples for 10 min, 95°C
Results
The gel does not show any enrichment of protein at 15 kDa
pIG15_1501 and pIG_1601: sonification
Experiment
- cell pellet (10ml sample) were resuspend in 5ml lysis buffer (20mM NaH2PO4, 300mM Nacl, 10mM Imidazole), cell pellet of the 500ml culture was resuspend in 10nml lysis buffer.
- cells were sonificated for 5min, with 6 cycles.
- after the sonification 1:1000 PMSF (stock 300mM) was added to each sample. PMSF is s protease inhibitor
- disrupted cells were centrifuged for 30 min, 10 000xg
- the supernatant was transferred in a new falcon and the pellet in the equal volume resuspended
- 9ul of each sample were taken for SDS-PAGE Analysis with 6ul of 2,5x SDS-Loading Dye
- for further analysis a 12% SDS-PAGE Gel was used
Results
in the 3h samples the target protein seems to be in the supernatant (in pIG15_1501 and pIG15_1601)
therefore both supernatant were used for the protein purification step
therefore both supernatant were used for the protein purification step
remark (02.06.15): pIG15_1601 contains the target protein only in the pellet. Further experiments have to be done.
protein purification pIG15_1501 and pIG1601
- 400ul slurry of the Ni-NTA agarose were transferred in a new falcon (cut tips!!)
- after centrifugation 2min, 500xg, the supernatant was discarded
- the beads were equlibrated with the lysis buffer
- after equlibration the supernatant was added, and incubated o/n, 4°C (keep the falcons rolling)
27.05.15
pIG15_803
Experiment
- o/n culture of transformed pIG15_803 8 in BL21 [psbc3 backbone + Herpes simplex] (Cml) for overexpression test. [picked several clones]
26.05.15
pIG15_1501 & pIG15_1601: 12%SDS-PAGE Analysis of overexpression
Experiment
- load 10 ul of each sample [before induction, 1h, 2h,3h, o/n] + 5µl prestained marker thermo scientifc
- stain gel with Coomassie staining solution for 30 min, wash gel with water and destain it with the „destain solution“
Results
pIG15_803 BL21 Transformation
Experiment
- pIG15_803 8 in BL21 [psbc3 backbone + Herpes simplex] (Cml)
- for overexpression test.
25.05.15
pIG15_1501 & pIG15_1601: overexpression
Experiments
- 500 ml LB-Medium Amp/Cml + o/n culture (5ml) [24.05.15]
- 37°C, 200 rpm
- grow until OD600 ~ 0.5
- take sample before induction
- add 500ul IPTG [final conc. 1mM]
- take every hour 20 ml sample after induction; 500ml sample after 3h → freeze pellets -20°C
- take also 1ml sample of each timepoint for SDS-PAGE Analysis, –> centrifuge at maximum speed, discard supernatant and add 200 of 2,5x SDS Loading Dye. Boil samples for 10 min, 95°C
- store at RT until SDS-PAGE Analysis
24.05.15
- 500 ml each (amp+cml) + o/n culture
→ wrong plasmids, new transformation with the right ones
Trafo:
- pIG15_1501 (amp) [salmonella in pet22a] in Rosetta (cml)
- His-tagged putative dihydroxyacid dehydratase (S. typhimurium)
- pIG15_1601 (amp) [salmonella in pet22a] in Rosetta (cml)
- His-tagged p dimethyl sulph reduct (S. typhimurium)
→ o/n culture (5ml) Amp/Cml of each pIG15_1501, pIG15_1601
23.05.15
- o/n culture 5 ml each (22.05.15)
22.05.15
Trafo
- pIG15_501 (amp) in Rosetta (cml)
- PIG15_601 (amp) in Rosetta (cml)
21.04.15
Trafo (Protocol)
- pET51b(+) [50ug/ul] 1ul in Rosetta (Cml) and Bl21 (Cml); testing competent cells –> cells are competent and do not have any contamination
