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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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
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
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
• 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!