Jul 12, 2019

Public workspaceHeterologous expression and affinity purification of Strep-tagged (KaiC) proteins

Journal of Bacteriology
DOI
dx.doi.org/10.17504/protocols.io.meac3ae
  • 1Heinrich-Heine Universität Düsseldorf;
  • 2Institute of Biology III, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;
  • 3Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
  • 4Institute for Microbiology and Molecular Biology, Justus-Liebig University, 35392 Giessen, Germany;
  • 5College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
  • 6Institute for Synthetic Microbiology, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University Duesseldorf, 40225 Duesseldorf, Germany
  • Axmann Lab
  • CyanoWorld
Anika Wiegard
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DOI: dx.doi.org/10.17504/protocols.io.meac3ae
External link: https://doi.org/10.1128/JB.00478-19
Protocol Citation: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria IM Axmann 2019. Heterologous expression and affinity purification of Strep-tagged (KaiC) proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.meac3ae
Manuscript citation:
Wiegard A, Köbler C, Oyama K, Dörrich AK, Azai C, Terauchi K, Wilde A, Axmann IM, Array. Journal of Bacteriology 202(4). doi: 10.1128/JB.00478-19
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 24, 2017
Last Modified: July 12, 2019
Protocol Integer ID: 9378
Keywords: pASK, Strep-tag, affinity chromatography, KaiC, recombinant protein, heterologous expression, cell lysis, protein purification
Abstract
This protocol can be used to:
(i) express Strep-tagged proteins in E.coli
(ii) lyse cells
(iii) purify Strep-tagged proteins via gravity flow affinity chromatography using either Strep-tactin XT superflow or Strep-Tactin resin
Materials
MATERIALS
ReagentNaOH
ReagentMagnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)
ReagentTris(hydroxymethyl)aminomethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859-500G
ReagentNaCl
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentBenzonaseMerck Millipore (EMD Millipore)Catalog #101654
ReagentRoche Complete Protease Inhibitor EDTA-Free tablets Merck MilliporeSigma (Sigma-Aldrich)Catalog #5056489001
ReagentAmpicillin sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0166
ReagentHCl
Reagent14 Dithiotreitol (DTT)Carl RothCatalog #6908.1
ReagentAdenosin-5-triphosphate disodium salt (ATP)Carl RothCatalog #HN35.1
ReagentQuick Start™ Bradford 1x Dye Reagent Bio-Rad LaboratoriesCatalog #5000205
ReagentLB broth (Lennox)Carl RothCatalog #X964.1
ReagentpASK-IBA5plusCatalog #2-1404-000
ReagentRosetta-gami™ B (DE3) Competent Cells - NovagenCatalog #71136
ReagentChloramphenicolCarl RothCatalog #3886.2
ReagentD( )BiotinCatalog #2-1016-002
ReagentAnhydrotetracyclineCatalog #2-0401-001
STEP MATERIALS
ReagentStrep-Tactin®XT Superflow® 50% suspensionCatalog #2-4010-010
ReagentStrep-Tactin® Sepharose® 50% suspensionCatalog #2-1201-010
ReagentD( )BiotinCatalog # 2-1016-002
Reagent10x Buffer R Strep-Tactin® Regeneration Buffer with HABA Catalog #2-1002-100
ReagentDesthiobiotinCatalog #2-1000-001

Protocol materials
ReagentLB broth (Lennox)Carl RothCatalog #X964.1
ReagentRosetta-gami™ B (DE3) Competent Cells - NovagenCatalog #71136
ReagentD( )BiotinCatalog # 2-1016-002
ReagentDesthiobiotinCatalog #2-1000-001
ReagentBenzonaseMerck Millipore (EMD Millipore)Catalog #101654
Reagent14 Dithiotreitol (DTT)Carl RothCatalog #6908.1
ReagentMagnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)
ReagentAdenosin-5-triphosphate disodium salt (ATP)Carl RothCatalog #HN35.1
ReagentQuick Start™ Bradford 1x Dye Reagent Bio-Rad LaboratoriesCatalog #5000205
ReagentStrep-Tactin® Sepharose® 50% suspensionCatalog #2-1201-010
ReagentTris(hydroxymethyl)aminomethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859-500G
ReagentAmpicillin sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0166
ReagentHCl
ReagentpASK-IBA5plusCatalog #2-1404-000
ReagentChloramphenicolCarl RothCatalog #3886.2
ReagentD( )BiotinCatalog #2-1016-002
ReagentAnhydrotetracyclineCatalog #2-0401-001
ReagentNaCl
ReagentNaOH
ReagentStrep-Tactin®XT Superflow® 50% suspensionCatalog #2-4010-010
Reagent10x Buffer R Strep-Tactin® Regeneration Buffer with HABA Catalog #2-1002-100
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentRoche Complete Protease Inhibitor EDTA-Free tablets Merck MilliporeSigma (Sigma-Aldrich)Catalog #5056489001
ReagentStrep-Tactin®XT Superflow® 50% suspensionCatalog #2-4010-010
ReagentStrep-Tactin® Sepharose® 50% suspensionCatalog #2-1201-010
ReagentD( )BiotinCatalog # 2-1016-002
ReagentDesthiobiotinCatalog #2-1000-001
Reagent10x Buffer R Strep-Tactin® Regeneration Buffer with HABA Catalog #2-1002-100
heterologous protein expression in E.coli
heterologous protein expression in E.coli


transformation
  • transform E.coli expression cells (e.g. Rosetta gamiB (DE3,) or Rosetta gami2 (DE3)) with your pASK based expression plasmid
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.
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 = ~0.5
Note: use erlenmeyer flasks with a volume of at least 4l to ensure sufficient aeration.
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
cell harvest
  • spin cells for 10 min at 4°C and 4000g
  • discard supernatant
  • keep cells on ice
Duration00:10:00 centrifugation
cell disruption
cell disruption
enzymatic lysis by lysozyme:
  • weight cell pellets
  • resuspend 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 benzonase
  • incubate on ice for 30 min
Note: you can also use 50 mM Tris in buffer
Note: you can skip enzymatic lysis step and perform longer sonication instead
Duration00:30:00 incubation on ice
sonication:
  • sonicate the cell suspension for 6 min on ice using e.g. a Bandelin sonopuls homogenizer and the following parameters
  • Note: During sonication, the temperature of the cell suspension should be kept below 15 °C
tip KE76
cycle 1 (0.1 sec active cycle, 0.9 sec passive cycle)
output 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)
Duration00:06:00 sonication
clarifaction of the lysate:
  • centrifuge the resulting lysate for 20 min at 4 °C and 23000 g to remove insolubles
  • keep 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.
Duration00:20:00 centrifugation
affinity purification
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 column
  • equilibrate with 30 ml ice-cold buffer W
  • alternatively: equilibrate with 25 ml ice-cold buffer W and subsequently with 5 ml ice-cold buffer W including protease inhibitor
Note: if resin was stored in buffer R before, the colour will change from orange to white
ReagentStrep-Tactin®XT Superflow® 50% suspensionCatalog #2-4010-010
ReagentStrep-Tactin® Sepharose® 50% suspensionCatalog #2-1201-010
protein binding (at 4°C or room temperature)
  • apply soluble proteins to your column
  • collect the flow though
washing (at 4°C or room temperature)
  • wash column with 15-50 ml ice-cold buffer W including protease inhibitors (at 4°C or room temperature)
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

ReagentD( )BiotinCatalog # 2-1016-002


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 desthiobiotin
  • collect fractions of 1 to 2ml
ReagentDesthiobiotinCatalog #2-1000-001

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 NaOH
  • optional: 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 regeneration
Note: you cannot use buffer R for regeneration of Strep-tactin XT superflow resin
Reagent10x Buffer R Strep-Tactin® Regeneration Buffer with HABA Catalog #2-1002-100
regeneration of Strep-Tactin Sepharose (if you use Strep-tactin XT superflow resin, you already finished regeneration with step 14) (at room temperature)
  • wash with buffer R (0.1 M Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM HABA (hydroxy-azophenyl-benzoic acid) until agarose turns orange
qualitative analysis of eluate fractions:
  • for each fraction, pipette 80 µl of Bradford solution in a well of a 96 well plate
  • add 5-20 µl of your fraction
  • colour change to blue indicates successful elution of proteins --> keep those fractions
  • control quality of eluted protein by separation via SDS-PAGE (optional: analyse aliquots of lysate, flow through and washing steps in parallel)
buffer exchange:
a) perform size exclusion chromatography or
b) exchange buffer using centrifugal concentrators
  • mix all eluate fractions of sufficient protein quality and transfer them to a disposable centrifugal concentrator
  • concentrate protein by centrifugation
  • add your desired buffer
  • concentrate again
  • repeat this step until the buffer is completely exchanged
Note: choose the molecular cut-off of the concentrator and centrifugal force according to the manufacturer’s instructions.