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

protein purification of pIG15_1301 (74kDa with gIII and 36kDa without gIII only VL and VH) by Ni-NTA beads, 12%SDS-PAGE Analysis

To confirm, whether the gIII (secretion signal to periplasm) is cleaved off, western blot should be performed with anti-His Tag antibdoy.

• transformation of the pET_Spy catcher in C43 competent cells (Amp+Cml)

• 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

• 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)

glycerol stock was done (shock freeze in liquid nitrogen, store @ -80°C)

• 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
• cell disruption protocol
• 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

12.5% SDS-PAGE Analysis. Cell disruption of C43 E.colistrain with the construct pIG15_1301

• anti-dehydroxyacid dehydratase is about 74kDa.
• the protein seems to soluble.
• some mistakes occur during the loaoding process of the samples

• 5ml o/n culture of C43 + pIG15_1301 for large scale over expression [Amp+Cml]

• 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

• 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 were performed according to the protocol competent cells

• 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

• 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]

• plasmids were obtained from AG Roth
• Transformation with Rosetta cells
• grown o/n on plate

• TOP10 + C43 (new expression strain from Toolbox) on plate with no and Cml, respectively.

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

• Cell lysis was performed according to cell disruption protocoll
• SDS-PAGE on 8.5 % Gel was performed according to SDS-PAGE protocol

• 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

• 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

• purification was performed as described in bead_purification
• beads were regenerated as described in the Ni-NTA bead regeneration protocol

Resulting protein concentration: blanked with elution buffer

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.

Purifcation of the pooled supernatants of 18°C 2h & 4h and 28°C 2h & 4h respectively. In every purification in total 20 mL of 100 mL culture were used. Gel 1 of 2.

Gel 2 of 2

After Elution with 500 mM imidazole there is protein visible at 65 kDa, the actual weight of the pIG15_1501 Salmonella-Antigen.

• started o/n culture of pIG15_1601 BL21 pLys in AMp/Cml

• 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

• 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

• 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

• streaked pIG15_1601 in Rosetta (Glycerolstock) on Cml/Amp plate → for further overespression experiments (testing several temperatures)

• SDS-PAGE with samples from 29.05.
• Coomassie-Staining for 20 min
• Destaining

Ni-NTA-beads were regenerated according to bead regeneration protocol.

• 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

protein purification of pIG15_1501 and pIG_1601. Elution was performed with 500mM Imidazole. FT=flow-through, W=wash; E=elution

protein concentration in eluate pIG15_1501

Using NanoDrop 2000c

protein concentration in eluate pIG15_1601

Using NanoDrop 2000c

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

• 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

• 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

sonification of cells: Rosetta with the construct pIG15_1501 and pIG1601, respectively. s=supernatant fraction, p=pellet fraction

remark (02.06.15): pIG15_1601 contains the target protein only in the pellet. Further experiments have to be done.

• 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)

• o/n culture of transformed pIG15_803 8 in BL21 [psbc3 backbone + Herpes simplex] (Cml) for overexpression test. [picked several clones]

• 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“

• pIG15_803 8 in BL21 [psbc3 backbone + Herpes simplex] (Cml)
• for overexpression test.

• 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

• 500 ml each (amp+cml) + o/n culture

→ wrong plasmids, new transformation with the right ones

• 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

• o/n culture 5 ml each (22.05.15)

• pIG15_501 (amp) in Rosetta (cml)
• PIG15_601 (amp) in Rosetta (cml)

• pET51b(+) [50ug/ul] 1ul in Rosetta (Cml) and Bl21 (Cml); testing competent cells –> cells are competent and do not have any contamination