TITLE: Heterologous expression and affinity purification of Strep-tagged (KaiC) 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 to:</div><div class = "text-block"><span>(i) express Strep-tagged proteins in </span><span style = "font-style:italic;">E.coli</span></div><div class = "text-block">(ii) lyse cells</div><div class = "text-block">(iii) purify Strep-tagged proteins via gravity flow affinity chromatography using either Strep-tactin XT superflow or Strep-Tactin resin</div></div>

[STEPS]
?. [heterologous protein expression in E.coli]
transformation
?. [heterologous protein expression in E.coli]
pre-culture:inoculate LB medium containing 75-100 µg ampicillin ml-1 (optional: plus 30 µg chloramphenicol ml-1) with resulting transformants (use e.g. 200 ml LB)incubate over night at 37 °C and 200-250 r.p.m.
?. [heterologous protein expression in E.coli]
expression culture:inoculate LB medium containing 75-100 µg ampicillin ml-1 (optional: plus 30 µg chloramphenicol ml-1) with 4-10 % volume of the pre-culture (add e.g. 200 ml pre-culture to 1.8 l LB)incubate at 37 °C and 200-250 r.p.m. until OD600nm = ~0.5Note: use erlenmeyer flasks with a volume of at least 4l to ensure sufficient aeration.
?. [heterologous protein expression in E.coli]
induction of protein expression:when cell density reaches OD600nm = ~0.3-0.7 (optimal 0.5) add 200 µl of 2 mg anhydrotetracycline ml-1 (final concentration 200 ng anhydrotetracycline ml-1)choose optimal expression condition (has to be tested for each protein of interest), e.g.: over night at 18-25 °C and 200-250 r.p.m. or at 35-37 °C and 200-250 r.p.m. for 3.5.-5.5 hours
?. [cell harvest]
spin cells for 10 min at 4°C and 4000gdiscard supernatantkeep cells on ice
[centrifugation]
?. [cell disruption]
enzymatic lysis by lysozyme:weight cell pelletsresuspend cells in ice-cold buffer W [20mM Tris/HCl (pH8), 150 mM NaCl, 2 mM DTT (only for KaiC proteins: 5 mM MgCl2, 1 mM ATP)] including protease inhibitors (e.g. protease inhibitor cocktail, Roche) using a paint brush. (use 3 ml buffer per g cells)add a spatula tip’s worth of lysozyme (or add lysozyme stock solution to a final concentration of 1 mg lysozyme ml-1)add 125 U benzonaseincubate on ice for 30 minNote: you can also use 50 mM Tris in bufferNote: you can skip enzymatic lysis step and perform longer sonication instead
[incubation on ice]
?. [cell disruption]
sonication:sonicate the cell suspension for 6 min on ice using e.g. a Bandelin sonopuls homogenizer and the following parametersNote: During sonication, the temperature of the cell suspension should be kept below 15 °C  AB1tip
KE76
2cycle
1 (0.1 sec active cycle, 0.9 sec passive cycle)
3output
60 %
Note: 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
1 (0.1 sec active cycle, 0.9 sec passive cycle)
3output
60 %
[sonication]
?. [cell disruption]
clarifaction of the lysate:centrifuge the resulting lysate for 20 min at 4 °C and 23000 g to remove insolubleskeep the resulting supernatant (= soluble proteins)Note: 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]
preparation of affinity column (at 4°C or room temperature)pour 3 ml Strep-tactin XT superflow resin (or Strep-Tactin Sepharose) in an appropriate glass columnequilibrate with 30 ml ice-cold buffer Walternatively: equilibrate with 25 ml ice-cold buffer W and subsequently with 5 ml ice-cold buffer W including protease inhibitorNote: if resin was stored in buffer R before, the colour will change from orange to white
?. [affinity purification]
protein binding (at 4°C or room temperature)apply soluble proteins to your columncollect the flow though
?. [affinity purification]
washing (at 4°C or room temperature)
?. [affinity purification]
elution from Strep-Tactin XT superflow resin (if you use Strep-Tactin Sepharose proceed with step 13 instead) (at 4°C or room temperature)elute proteins with 9 ml ice cold buffer BXT (buffer W + 50 mM D(+)biotin)collect 6 fractions of 1.5 to 2ml
?. [affinity purification]
elution from Strep-Tactin Sepharose (if you use Strep-tactin XT superflow resin skip this step and move on to step 14) (at 4°C or room temperature)elute proteins with 15-30 ml ice cold buffer W + 2.5 mM desthiobiotincollect fractions of 1 to 2ml
?. [affinity purification]
regeneration of Strep-Tactin XT superflow resin (if you use Strep-Tactin Sepharose proceed with step 15 instead) (at room temperature)wash with 15 ml 10 mM freshly prepared NaOHoptional: regeneration can be tested by adding buffer R (100 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM HABA (hydroxy-azophenyl-benzoic acid). An orange-shift indicates successful regenerationNote: you cannot use buffer R for regeneration of Strep-tactin XT superflow resin
?. [affinity purification]
regeneration of Strep-Tactin Sepharose (if you use Strep-tactin XT superflow resin, you already finished regeneration with step 14) (at room temperature)
?. [affinity purification]
qualitative analysis of eluate fractions:for each fraction, pipette 80 µl of Bradford solution in a well of a 96 well plateadd 5-20 µl of your fractioncolour change to blue indicates successful elution of proteins --> keep those fractionscontrol quality of eluted protein by separation via SDS-PAGE (optional: analyse aliquots of lysate, flow through and washing steps in parallel)
?. [affinity purification]
buffer exchange:a) perform size exclusion chromatography orb) exchange buffer using centrifugal concentratorsmix all eluate fractions of sufficient protein quality and transfer them to a disposable centrifugal concentratorconcentrate protein by centrifugationadd your desired bufferconcentrate 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.