Luscher Lab Meeting

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Expression and Purification of Expression and Purification of His His 6 6 -NLS-Cre-MTS Protein from -NLS-Cre-MTS Protein from E. E. coli coli BL21(DE3) BL21(DE3) Heather Jordan Heather Jordan Department of Biochemistry and Molecular Biology Department of Biochemistry and Molecular Biology The Pennsylvania State University The Pennsylvania State University University Park, Pennsylvania 16802 University Park, Pennsylvania 16802 December 10, 2002 December 10, 2002 http://www.ozfactors.com/PICTURES /PSYCHS/Psychiatry.html

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Transcript of Luscher Lab Meeting

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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

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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)

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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

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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

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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

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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

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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

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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

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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.

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How does Cre work?How does Cre work?• Model of Recombination:Model of Recombination:

Strand swapping occurs and the Holliday junction intermediate is formed.

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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.

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How does Cre work?How does Cre work?• Model of Recombination:Model of Recombination:

Modified from http://www.esb.utexas.edu/jayaramlab/model.html

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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

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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

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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

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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

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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

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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?

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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:

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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:

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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:

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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:

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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:

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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))

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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

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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)

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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

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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)

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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!