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Expression and Purification of HisExpression and Purification of His66-NLS--NLS-
Cre-MTS Protein from Cre-MTS Protein from E. coliE. coli BL21(DE3) BL21(DE3)
Heather JordanHeather Jordan
Department of Biochemistry and Molecular BiologyDepartment of Biochemistry and Molecular Biology
The Pennsylvania State UniversityThe Pennsylvania State University
University Park, Pennsylvania 16802University Park, Pennsylvania 16802
December 10, 2002December 10, 2002
http://www.ozfactors.com/PICTURES/PSYCHS/Psychiatry.html
What is What is HisHis66--NLSNLS-Cre--Cre-MTSMTS??• A recombinant cell-permeable Cre proteinA recombinant cell-permeable Cre protein
1)1) HisHis66 affinity tag affinity tag• Binds to resin Binds to resin (part of purification process)(part of purification process)
2)2) Nuclear Localization SignalNuclear Localization Signal• Located anywhere in the primary sequence of the protein Located anywhere in the primary sequence of the protein • Directs the protein for transport through the nuclear pore complex Directs the protein for transport through the nuclear pore complex
(from cytosol to nucleus)(from cytosol to nucleus)
3)3) Membrane Translocation SequenceMembrane Translocation Sequence• Directs protein into the cell Directs protein into the cell (from cell surface to cytosol)(from cell surface to cytosol)
Cre protein is…Cre protein is…• A member of the A member of the integraseintegrase family family (catalyzes an insertion reaction in (catalyzes an insertion reaction in
DNA)DNA)
• Encoded by the Encoded by the E. coliE. coli phage phage P1P1
• During cell division, Cre During cell division, Cre separates dimersseparates dimers of P1 that arise after of P1 that arise after replication replication
Cre protein is…Cre protein is…• A member of the A member of the integraseintegrase family family (catalyzes an insertion reaction in (catalyzes an insertion reaction in
DNA)DNA)
• Encoded by the Encoded by the E. coliE. coli phage phage P1P1
• During cell division, Cre During cell division, Cre separates dimersseparates dimers of P1 that arise after of P1 that arise after replication replication
Cre protein is…Cre protein is…• A member of the A member of the integraseintegrase family family (catalyzes an insertion reaction in (catalyzes an insertion reaction in
DNA)DNA)
• Encoded by the Encoded by the E. coliE. coli phage phage P1P1
• During cell division, Cre During cell division, Cre separates dimersseparates dimers of P1 that arise after of P1 that arise after replication replication
ReplicationReplication
Cre protein is…Cre protein is…• A member of the A member of the integraseintegrase family family (catalyzes an insertion reaction in (catalyzes an insertion reaction in
DNA)DNA)
• Encoded by the Encoded by the E. coliE. coli phage phage P1P1
• During cell division, Cre During cell division, Cre separates dimersseparates dimers of P1 that arise after of P1 that arise after replication replication
Homologous Homologous RecombinationRecombination
ReplicationReplication
Cre protein is…Cre protein is…• A member of the A member of the integraseintegrase family family (catalyzes an insertion reaction in (catalyzes an insertion reaction in
DNA)DNA)
• Encoded by the Encoded by the E. coliE. coli phage phage P1P1
• During cell division, Cre During cell division, Cre separates dimersseparates dimers of P1 that arise after of P1 that arise after replication replication
Cre-mediated Cre-mediated recombinationrecombination
Homologous Homologous RecombinationRecombination
ReplicationReplication
Modified fromModified from http://www.esb.utexas.http://www.esb.utexas.edu/jayaramlab/recomedu/jayaramlab/recomb/Cre.htmlb/Cre.html
How does Cre work?How does Cre work?
• Site of action is the Site of action is the loxPloxP site. site.• loxPloxP consists of two consists of two 13 bp inverted13 bp inverted binding sitesbinding sites
separated by a separated by a 6 bp6 bp spacerspacer..• Cleavage sitesCleavage sites
loxP siteloxP site
How does Cre work?How does Cre work?• Model of Recombination:Model of Recombination:
Cre reacts with a 6 bp spacer and cleaves in cis at the arrowheads. Proteins bring the DNA site together in an antiparallel synapse with the green monomers active and the blue monomers supressed.
How does Cre work?How does Cre work?• Model of Recombination:Model of Recombination:
Strand swapping occurs and the Holliday junction intermediate is formed.
How does Cre work?How does Cre work?• Model of Recombination:Model of Recombination:
The proteins undergo an allosteric conformational change. Now, the blue monomers are active for cleavage and the green ones are supressed.
How does Cre work?How does Cre work?• Model of Recombination:Model of Recombination:
Modified from http://www.esb.utexas.edu/jayaramlab/model.html
Study ObjectiveStudy Objective• In floxed In floxed (fg2)(fg2) mice the mice the
addition of the protein addition of the protein pops out the gene pops out the gene coding for the function coding for the function of axon 8 in the of axon 8 in the -2 -2 subunit.subunit.
• Can be used Can be used selectively to knock out selectively to knock out this gene in different this gene in different regions of the brainregions of the brain
http://web1.nsac.ns.ca/news/ac_post/1997/aug97/mouse.htm
Why knock out Why knock out -2 this way?-2 this way?
Current Method:Current Method: Cross-breedingCross-breeding
New Method:New Method: Protein InjectionProtein Injection
EMX-1
Materials and MethodsMaterials and MethodsI.I. ExpressionExpression
• Growing the Growing the E. coliE. coli• Induce over expression with IPTGInduce over expression with IPTG• Freezing the cellsFreezing the cells
II.II. PurificationPurification• Sonication & centrifugationSonication & centrifugation• Ni-NTA resin & elutionNi-NTA resin & elution
III.III. PreparationPreparation• FilterFilter• DialysisDialysis
• With aCSFWith aCSF
• QuantitationQuantitation• Bradford AssayBradford Assay
Materials and MethodsMaterials and MethodsI.I. ExpressionExpression
• Growing the Growing the E. coliE. coli• Induce over expression with IPTGInduce over expression with IPTG• Freezing the cellsFreezing the cells
II.II. PurificationPurification• Sonication & centrifugationSonication & centrifugation• Ni-NTA resin & elutionNi-NTA resin & elution
III.III. PreparationPreparation• FilterFilter• DialysisDialysis
With With aCSFaCSF
• QuantitationQuantitation Bradford AssayBradford Assay
Precipitate?!Precipitate?!That’s not supposed to be there!That’s not supposed to be there!
Why?Why?• High [salt]High [salt]
• pHpH
How do we find out what’s How do we find out what’s wrong?wrong?
Recalculated protocol Recalculated protocol amounts amounts (esp. hydrated salts)(esp. hydrated salts)
pH individual components of pH individual components of aCSFaCSF
Test every 2 components Test every 2 components (of (of the 3)the 3) against each other to against each other to see if ppt. formssee if ppt. forms
Results of 2-Component Results of 2-Component TestTest
aCSF alone = CLEARaCSF alone = CLEAR
aCSF + MgCL2 = CLEARaCSF + MgCL2 = CLEAR
aCSF + CaCl2 =aCSF + CaCl2 = WHITE PPT.WHITE PPT.
Is there another protocol for aCSF that might work better?Is there another protocol for aCSF that might work better?
Artificial Cerebrospinal Fluid Artificial Cerebrospinal Fluid FormulationsFormulations
• NaCl (124 mM)NaCl (124 mM)
• KCl (1.6 mM)KCl (1.6 mM)
• GlucoseGlucose
• CaClCaCl22 (2.5 mM) (2.5 mM)
• MgClMgCl22 (1.5 mM) (1.5 mM)
• NaHCONaHCO33 (24 mM) (24 mM)
• KHKH22POPO44 (1.2 mM) (1.2 mM)
• Ascorbic Acid (2 mM)Ascorbic Acid (2 mM)
• NaCl (147 mM)NaCl (147 mM)
• KCl (2.9 mM)KCl (2.9 mM)
• DextroseDextrose
• CaClCaCl22 (1.7 mM) (1.7 mM)
• MgClMgCl22 (1.6 mM) (1.6 mM)
First, we tried:First, we tried: Then, we tried:Then, we tried:
Artificial Cerebrospinal Fluid Artificial Cerebrospinal Fluid FormulationsFormulations
• NaCl (NaCl (124 mM124 mM))
• KCl (KCl (1.6 mM1.6 mM))
• GlucoseGlucose
• CaClCaCl22 ( (2.5 mM2.5 mM))
• MgClMgCl22 ( (1.5 mM1.5 mM))
• NaHCONaHCO33 (24 mM) (24 mM)
• KHKH22POPO44 (1.2 mM) (1.2 mM)
• Ascorbic Acid (2 mM)Ascorbic Acid (2 mM)
• NaCl (NaCl (147 mM147 mM))
• KCl (KCl (2.9 mM2.9 mM))
• DextroseDextrose
• CaClCaCl22 ( (1.7 mM1.7 mM))
• MgClMgCl22 ( (1.6 mM1.6 mM))
First, we tried:First, we tried: Then, we tried:Then, we tried:
Artificial Cerebrospinal Fluid Artificial Cerebrospinal Fluid FormulationsFormulations
• NaCl (NaCl (124 mM124 mM))
• KCl (KCl (1.6 mM1.6 mM))
• GlucoseGlucose
• CaClCaCl22 ( (2.5 mM2.5 mM))
• MgClMgCl22 ( (1.5 mM1.5 mM))
• NaHCONaHCO33 (24 mM) (24 mM)
• KHKH22POPO44 (1.2 mM) (1.2 mM)
• Ascorbic Acid (2 mM)Ascorbic Acid (2 mM)
• NaCl (NaCl (147 mM147 mM))
• KCl (KCl (2.9 mM2.9 mM))
• DextroseDextrose
• CaClCaCl22 ( (1.7 mM1.7 mM))
• MgClMgCl22 ( (1.6 mM1.6 mM))
First, we tried:First, we tried: Then, we tried:Then, we tried:
Artificial Cerebrospinal Fluid Artificial Cerebrospinal Fluid FormulationsFormulations
• NaCl (NaCl (124 mM124 mM))
• KCl (KCl (1.6 mM1.6 mM))
• GlucoseGlucose
• CaClCaCl22 ( (2.5 mM2.5 mM))
• MgClMgCl22 ( (1.5 mM1.5 mM))
• NaHCONaHCO33 (24 mM) (24 mM)
• KHKH22POPO44 (1.2 mM) (1.2 mM)
• Ascorbic Acid (2 mM)Ascorbic Acid (2 mM)
• NaCl (NaCl (147 mM147 mM))
• KCl (KCl (2.9 mM2.9 mM))
• DextroseDextrose
• CaClCaCl22 ( (1.7 mM1.7 mM))
• MgClMgCl22 ( (1.6 mM1.6 mM))
First, we tried:First, we tried: Then, we tried:Then, we tried:
Artificial Cerebrospinal Fluid Artificial Cerebrospinal Fluid FormulationsFormulations
• NaCl (124 mM)NaCl (124 mM)
• KCl (1.6 mM)KCl (1.6 mM)
• GlucoseGlucose
• CaClCaCl22 (2.5 mM) (2.5 mM)
• MgClMgCl22 (1.5 mM) (1.5 mM)
• NaHCONaHCO33 (24 mM) (24 mM)
• KHKH22POPO44 (1.2 mM) (1.2 mM)
• Ascorbic Acid (2 mM)Ascorbic Acid (2 mM)
• NaCl (NaCl (147 mM147 mM))
• KCl (KCl (2.9 mM2.9 mM))
• DextroseDextrose
• CaClCaCl22 ( (1.7 mM1.7 mM))
• MgClMgCl22 ( (1.6 mM1.6 mM))
First, we tried:First, we tried: Then, we tried:Then, we tried:
How was this conclusion reached?How was this conclusion reached?
• Had the same problem Had the same problem making anaerobic making anaerobic mediamedia
• The The NaHCONaHCO33 was was
identified as the identified as the ‘problem ingredient’‘problem ingredient’
• Also yielded Also yielded white white precipitateprecipitate
• New buffer system New buffer system devised (using devised (using KHKH22POPO44
and Kand K22HPOHPO44 instead of instead of
NaHCONaHCO33))
Other Problems EncounteredOther Problems Encountered
• Shaker Shaker temperaturetemperature – Changed from 37Changed from 37ooC C
to 25to 25ooC.C.
• Centrifuge noiseCentrifuge noise– Tubes not swinging Tubes not swinging
freelyfreely– Slight imbalanceSlight imbalance
ResultsResults
Mol. Wt. vs Distance Traveled (SDS-PAGE)
y = 75.148x-1.1081
0
50
100
150
200
250
300
0 1 2 3 4 5
Distance Traveled (cm)
Mo
l. W
t. (
kDa)
Series1
Power (Series1)
ResultsResults
• Band ABand A: 75.148x: 75.148x-1.1081-1.1081 = = 1.31.3 x = x = 38.9 kDa38.9 kDa• Band BBand B: 75.148x: 75.148x-1.1081-1.1081 = = 2.32.3 x = x = 23.25 kDa23.25 kDa• Band CBand C: 75.148x: 75.148x-1.1081-1.1081 = = 2.72.7 x = x = 20.12 kDa20.12 kDa
Desired Desired Protein Protein ProductProduct
Possible Possible Protein Protein Degradation Degradation ProductsProducts
AA
BB
CC
Ladder Ladder Pst-sonPst-son Pst-B2 Pst-B2 Pst-B3Pst-B3 Flo-Th Flo-Th FinalFinal
1.31.3
2.32.3
2.72.7
Future DirectionsFuture Directions
• Most protein loss occurred early in Most protein loss occurred early in protocolprotocol (before first aliquot)(before first aliquot)
• Try it again with the following changes:Try it again with the following changes:– Less sonication Less sonication (extra bands are probably (extra bands are probably
degradation products of desired protein)degradation products of desired protein)– Monitor sonication more closely Monitor sonication more closely (checking (checking
viscosity)viscosity)
• Leave purification steps unchanged Leave purification steps unchanged (no (no significant contamination in aliquots)significant contamination in aliquots)
Acknowledgements
• Clint EarnheartClint Earnheart
• Everyone else in Everyone else in the Lthe Lüüscher labscher lab
http://www.criver.com/xruk/transgenics.html
- I’m - I’m chimeric chimeric but I’m but I’m cute!cute!