TITLE: Expression and purification of (GST-tagged) (Kai) proteins
AUTHORS: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used for:</div><div class = "text-block"><span>(i) heterologous expression of GST-tagged proteins from pGEX-6P1 based expression vectors in </span><span style = "font-style:italic;">E. coli</span><span style = "font-style:italic;">.</span></div><div class = "text-block">(ii) purification of recombinant proteins via affinity chromatography using glutathione-agarose or glutathione-sepharose (GST tagged protein can be eluted with glutathione. Alternatively, the tag can be cleaved off by prescission protease)</div><div class = "text-block">(iii) further purification of the eluted protein via anion exchange chromatography</div><div class = "text-block">This protocol was modified from</div><div class = "text-block"><span>Wiegard A, Dörrich AK, Deinzer HT, Beck C, Wilde A, Holtzendorff J, Axmann IM: Biochemical analysis of three putative KaiC clock proteins from </span><span style = "font-style:italic;">Synechocystis </span><span>sp. PCC 6803 suggests their functional divergence. </span><span style = "font-style:italic;">Microbiology </span><span>2013, 159, 948-958</span></div><div class = "text-block"><span>Snijder J, Schuller JM, Wiegard A, Lössel, P, Schmelling NM, Axmann IM, Plitzko JM, Förster F, Heck AJR: Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. </span><span style = "font-style:italic;">Science</span><span> 2017, 355(6330):1181-1184</span></div></div>

[STEPS]
?. [heterologous protein expression in E.coli]
transformation:
?. [heterologous protein expression in E.coli]
pre-culture:inoculate 100 ml terrific broth medium containing 100 µg ampicillin ml-1 with resulting transformantsincubate over night at 37 °C and 200-250 r.p.m.
?. [heterologous protein expression in E.coli]
expression culture:inoculate 1 l terrific broth medium containing 100 µg ampicillin ml-1 with the pre-cultureincubate at 37 °C and 200-250 r.p.m.Note: use erlenmeyer flasks with a volume of at least 2 l to ensure sufficient aeration.
?. [heterologous protein expression in E.coli]
induction of protein expression:For KaiC proteins:grow cells without induction at 37 °C for approx. 72 hours Note: the promotor is leaky and will allow low protein expression and production of soluble protein, even without induction. Contrary to this, induction of kaiC expression leads to protein aggregation in inclusion bodies. For KaiA and KaiB proteins:grow cells for 3 hours at 37 °Cadd IPTG to a final concentration of 1 mM andcontinue incubation at 37 °C and 200-250 r.p.m. over nightNote: the promotor is leaky and will allow low protein expression and production of soluble protein, even without induction. Contrary to this, induction of kaiC expression leads to protein aggregation in inclusion bodies.For KaiA and KaiB proteins:grow cells for 3 hours at 37 °Cadd IPTG to a final concentration of 1 mM andcontinue incubation at 37 °C and 200-250 r.p.m. over night
?. [cell harvest]
spin down cells for 10 min at 4°C and 4000gdiscard supernatant and keep cells on ice.
[centrifugation]
?. [cell disruption]
enzymatic lysis by lysozyme:resuspend cells in 15 ml ice-cold extraction buffer [50 mM Tris/HCl (pH8), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT (only for KaiC proteins: 5 mM MgCl2, 1 mM ATP)] . For this resuspend pellet thoroughly in 5 ml buffer using a paint brush or pasteur pipette. Then add the remaining 10 ml buffer and mix well without creatung foamadd a spatula tip’s worth of lysozyme (or add lysozyme stock solution to a final concentration of 1mg lysozyme ml-1)add 125 U benzonaseincubate on ice for 30 min. Optional: mix every 5 min or place on a magnetic stirrerNote: you can skip enzymatic lysis step and perform longer sonication instead
[incubation on ice]
?. [cell disruption]
sonication:Note: During sonication, the temperature of the cell suspension should be kept below 15 °CNote: if you did not perform enzymatic lysis as described before, use alternative prolonged sonication conditions (e.g. alternation of 10 sec pulse and 10 sec pause for 25 minutes with 30 % output)  AB1tip
KE76
2cycle
3 (0.3 sec active cycle, 0.7 sec passive cycle)
3output
60 %
AB1tip
KE76
2cycle
3 (0.3 sec active cycle, 0.7 sec passive cycle)
3output
60 %
[sonication]
?. [cell disruption]
clarification of the lysate:centrifuge the resulting lysate for 20 min at 4 °C and 23000 g to remove insolublesKeep the resulting supernatant (= soluble proteins) on iceNote: Many conical centrifugation tubes cannot withstand centrifugation of 23000g. If you want to use them, you can reduce centrifugal force, while increasing centrifugation time.
[centrifugation]
?. [affinity purification]
equilibration of glutathione resin:swirl stock solution of glutathione agarose 4B or glutathione sepharose 4B gentlytransfer 1 ml in a 50 ml conical centrifugation tubeAdd 5-10 ml extraction buffercentrifuge for 4 min at 1500 g and 4 °C using a swing out rotordiscard supernatant carefully.
[centrifugation]
?. [affinity purification]
protein binding:add soluble proteins from step 8 to glutathione resinrotate overhead for at least 20 min at RT (use e.g. an intelli mixer and program F1 at 5 r.p.m).Note: incubation can be extended up to 3 hours.
[incubation at RT]
?. [affinity purification]
spin down glutathione resin for 4 min at 1500 g and 4 °C using a swing out rotordiscard supernatant carefully.
[centrifugation]
?. [affinity purification]
washing step 1:add 45 ml ice cold extraction buffermix thoroughlycentrifuge for 4 min at 1500 g and 4 °C using a swing out rotordiscard supernatant carefully.
[centrifugation]
?. [affinity purification]
washing step 2:add 45 ml ice cold extraction buffermix thoroughlycentrifuge for 4 min at 1500 g and 4 °C using a swing out rotordiscard supernatant carefully.
[centrifugation]
?. [affinity purification]
washing step 3:add 45 ml ice cold extraction buffermix thoroughlycentrifuge for 4 min at 1500 g and 4 °C using a swing out rotordiscard supernatant carefully.
[centrifugation]
?. [affinity purification]
washing step 4:add 45 ml ice cold extraction buffermix thoroughlycentrifuge for 4 min at 1500 g and 4 °C using a swing out rotordiscard supernatant carefully.
[centrifugation]
?. [affinity purification]
use 1 ml ice cold prescission buffer [50 mM Tris/HCl (pH8), 150 mM NaCl, 1 mM EDTA, 1 mM DTT (only for KaiC proteins: 5 mM MgCl2, 1 mM ATP)] to transfer pelleted resin to a 2 ml reaction tubecentrifuge for 4 min at 1500 g and 4 °Cdiscard supernatant using a pipetterepeat this step until all of glutathione resin is transferred to the reaction tube.Note: if you want to elute the GST-fused protein later (without cleavage by prescission protease), you can use extraction buffer instead of prescission buffer.
[centrifugation]
?. [option 1: elution of the untagged protein (part of affinity purification)]
overnight cleavage:mix glutathione resin with 500 µl ice cold prescission bufferadd 12.5 µl prescission proteaseincubate overnight at 4 °C under constant rotation of the tube (use e.g. an intelli mixer and program F4 at 5 r.p.m).Note: This section describes elution of the untagged protein via cleavage by prescission protease. If you want to elute the recombinant GST-tagged protein (without removal of the tag), skip these steps and move on to step 23 instead.
[centrifugation]
?. [option 1: elution of the untagged protein (part of affinity purification)]
elution 1:spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 1).Note: This section describes elution of the untagged protein via cleavage by prescission protease. If you want to elute the recombinant GST-tagged protein (without removal of the tag), skip these steps and move on to step 23 instead.
[centrifugation]
?. [option 1: elution of the untagged protein (part of affinity purification)]
elution 2:spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 2).Note: This section describes elution of the untagged protein via cleavage by prescission protease. If you want to elute the recombinant GST-tagged protein (without removal of the tag), skip these steps and move on to step 23 instead.
[centrifugation]
?. [option 1: elution of the untagged protein (part of affinity purification)]
elution 3:spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 3).Note: This section describes elution of the untagged protein via cleavage by prescission protease. If you want to elute the recombinant GST-tagged protein (without removal of the tag), skip these steps and move on to step 24 instead.
[centrifugation]
?. [option 1: elution of the untagged protein (part of affinity purification)]
elution 4:spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 4).Note: This section describes elution of the untagged protein via cleavage by prescission protease. If you want to elute the recombinant GST-tagged protein (without removal of the tag), skip these steps and move on to step 23 instead.
[centrifugation]
?. [option 1: elution of the untagged protein (part of affinity purification)]
elution 5:spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 5).Note: This section describes elution of the untagged protein via cleavage by prescission protease. If you want to elute the recombinant GST-tagged protein (without removal of the tag), skip these steps and move on to step 23 instead.
[centrifugation]
?. [option 2: elution of the GST-tagged protein (part of affinity purification)]
elution 1:freshly prepare glutathione solution [weight 40 mM reduced L-glutathion and 50 mM Tris, dissolve in water and adjust to pH 8 with NaOH (important!);  only for KaiC: add 5 mM MgCl2, 1 mM ATP]add 1 ml glutathione solution incubate for 5 min at RT under constant rotation of the tube (use e.g. an intelli mixer and program F4 at 5 r.p.m)spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 1).Note: This section describes the elution of the GST-tagged protein. If you removed the GST-tag by cleavage with prescission (steps 17-22), you have to skip these steps. Please move on to step 28 instead.
[incubation]
[centrifugation]
?. [option 2: elution of the GST-tagged protein (part of affinity purification)]
elution 2:Add 500 µl glutathione solutionincubate for 5 min at RT under constant rotation of the tube (use e.g. an intelli mixer and program F4 at 5 r.p.m)spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 2).Note: This section describes the elution of the GST-tagged protein. If you removed the GST-tag by cleavage with prescission (steps 17-22), you have to skip these steps. Please move on to step 28 instead.
[incubation]
[centrifugation]
?. [option 2: elution of the GST-tagged protein (part of affinity purification)]
elution 3:add 500 µl glutathione solution and incubate for 5 min at RT under constant rotation of the tube (use e.g. an intelli mixer and program F4 at 5 r.p.m)spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 3).Note: This section describes the elution of the GST-tagged protein. If you removed the GST-tag by cleavage with prescission (steps 17-22), you have to skip these steps. Please move on to step 28 instead.
[incubation]
[centrifugation]
?. [option 2: elution of the GST-tagged protein (part of affinity purification)]
elution 4:add 500 µl glutathione solution and incubate for 5 min at RT under constant rotation of the tube (use e.g. an intelli mixer and program F4 at 5 r.p.m)spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 4).Note: This section describes the elution of the GST-tagged protein. If you removed the GST-tag by cleavage with prescission (steps 17-22), you have to skip these steps. Please move on to step 28 instead.
[incubation]
[centrifugation]
?. [option 2: elution of the GST-tagged protein (part of affinity purification)]
elution 5:add 500 µl glutathione solution and incubate for 5 min at RT under constant rotation of the tube (use e.g. an intelli mixer and program F4 at 5 r.p.m)spin down glutathione resin for 4 min at 1500 g and 4 °Ctransfer the supernatant to a fresh tube (=eluate 5).Note: This section describes the elution of the GST-tagged protein. If you removed the GST-tag by cleavage with prescission (steps 17-22), you have to skip these steps. Please move on to step 28 instead.
[incubation]
[centrifugation]
?. [affinity purification]
qualitative analysis of elutate fractions:for each fraction, mix 80 µl of Bradford solution with 5-20 µl of your fraction in a well of a 96 well platea colour change to blue indicates sucessfull elution of proteinskeep those fractions.Note: You can further control quality of the protein by separation via SDS-PAGE.
?. [affinity purification]
buffer exchange:mix all eluate fractions of sufficient protein qualitytransfer them to a disposable centrifugal concentratorconcentrate protein by centrifugationadd your desired buffer and concentrate againrepeat this step until the buffer is completely exchangedNote: choose the molecular cut-off of the concentrator and centrifugal force according to the manufacturer’s instructions. For Kai proteins you can use the following buffers (depending on the desired application):  AB1KaiA
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
2KaiB
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
3KaiC
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 1mM ATP
Note: It is important to remove glutathione by sufficient buffer exchange. If you eluted the protein in prescission buffer and want to further purify it via anion exchange chromatography, buffer exchange is not necessary. You can control homogeneity of your protein by separation via SDS-PAGE. If it is sufficient for your application, you can stop the protocol here. If it is not sufficient for your application, you have to further purify your protein via anion exchange chromatography (steps 30-35)
AB1KaiA
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
2KaiB
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
3KaiC
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 1mM ATP
?. [anion exchange chromatography]
set-up of your liquid chromatography system:connect 10 ml sample loop to your systemconnect a MonoQ column (1 ml) or ResourceQ column (1 ml) to your systemexchange ethanol in capillaries and sample loop by waterwash the column with at least 10 column volumes degassed MilliQ (do not use higher flowrates than 1 min/ml)connect the following buffers as eluant A and eluent B:  AB1eluent
buffer composition
2A
50 mM Tris/HCl (pH8), 1 mM EDTA, 1 mM DTT
only for KaiC proteins: 5 mM MgCl2, 1 mM ATP
3B
50 mM Tris/HCl (pH8), 1 M NaCl, 1 mM EDTA, 1 mM DTT
only for KaiC proteins: 5 mM MgCl2, 1 mM ATP
AB1eluent
buffer composition
2A
50 mM Tris/HCl (pH8), 1 mM EDTA, 1 mM DTT
only for KaiC proteins: 5 mM MgCl2, 1 mM ATP
3B
50 mM Tris/HCl (pH8), 1 M NaCl, 1 mM EDTA, 1 mM DTT
only for KaiC proteins: 5 mM MgCl2, 1 mM ATP
?. [anion exchange chromatography]
equilibration of the column:Note: Do not use higher flowrates than 1 ml/min. It is better to equilibrate at lower flow rates overnight.
?. [anion exchange chromatography]
optional:if you want to test your set-up you can run the following program without injecting a samplemonitor absorption at 280 nm  ABCDE1column volume
flowrate
&nbsp;eluent
action
fractions
210
1 ml/min
100 % A
autozero
no
32
1 ml/min
100 % A
&nbsp;
no
420
1 ml/min
gradient from 100 % A to 100 % B
&nbsp;
no
55
1 ml/min
100 % B
&nbsp;
no
65
1 ml/min
100 A
&nbsp;
no
Note: If your instrument can measure two wavelengths in parallel, monitor absorption at 280 and 220 nm
ABCDE1column volume
flowrate
&nbsp;eluent
action
fractions
210
1 ml/min
100 % A
autozero
no
32
1 ml/min
100 % A
&nbsp;
no
420
1 ml/min
gradient from 100 % A to 100 % B
&nbsp;
no
55
1 ml/min
100 % B
&nbsp;
no
65
1 ml/min
100 A
&nbsp;
no
?. [anion exchange chromatography]
protein purification:dilute your protein in 10 ml eluent A and apply it to the sample loop (injection valve must be set to load position)run the following programmonitor absorption at 280 nmcollect fractions of 0.5 ml or 1 ml.  ABCDE1column volume
flowrate
&nbsp;eluent
action
fractions
210
1 ml/min
100 % A
autozero
inject position
0.5-1ml
32
1 ml/min
100 % A
load position
0.5-1ml
420
1 ml/min
gradient from 100 % A to 100 % B
&nbsp;
0.5-1ml
55
1 ml/min
100 % B
&nbsp;
0.5-1ml
65
1 ml/min
100 % A
&nbsp;
0.5-1ml
Note: If your instrument can measure two wavelengths in parallel, monitor absorption at 280 and 220 nm
ABCDE1column volume
flowrate
&nbsp;eluent
action
fractions
210
1 ml/min
100 % A
autozero
inject position
0.5-1ml
32
1 ml/min
100 % A
load position
0.5-1ml
420
1 ml/min
gradient from 100 % A to 100 % B
&nbsp;
0.5-1ml
55
1 ml/min
100 % B
&nbsp;
0.5-1ml
65
1 ml/min
100 % A
&nbsp;
0.5-1ml
?. [anion exchange chromatography]
qualitative analysis of eluate fractions:choose fractions of interest based on the absorption at 280 nmmix 5-20 µl of each fraction of interest with 80 µl of Bradford solution in a well of a 96 well platea colour change to blue indicates that you successfully eluted proteinskeep those fractionscontrol homogeneity and size of your eluted protein by separation via SDS-PAGE.
?. [anion exchange chromatography]
buffer exchange:concentrate your protein by centrifugationadd your desired bufferconcentrate againrepeat this step until buffer is completely exchangedNote: choose the molecular cut-off of the concentrator and centrifugal force according to the manufacturer’s instructions.For Kai proteins you can use the following buffers (depending on your desired application):  AB1KaiA
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
2KaiB
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
3KaiC
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 1mM ATP
AB1KaiA
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
2KaiB
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA (optional 5 mM MgCl2, 1mM ATP)
3KaiC
20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 1mM ATP