Luscher Lab Meeting

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  • 1. Expression and Purification of His 6 -NLS-Cre-MTS Protein from E. coli BL21(DE3) Heather Jordan Department of Biochemistry and Molecular Biology The Pennsylvania State University University Park, Pennsylvania 16802 December 10, 2002 http://www.ozfactors.com/PICTURES/PSYCHS/Psychiatry.html
  • 2. What is His 6 - NLS -Cre- MTS ?
    • A recombinant cell-permeable Cre protein
      • His 6 affinity tag
        • Binds to resin (part of purification process)
      • Nuclear Localization Signal
        • Located anywhere in the primary sequence of the protein
        • Directs the protein for transport through the nuclear pore complex (from cytosol to nucleus)
      • Membrane Translocation Sequence
        • Directs protein into the cell (from cell surface to cytosol)
  • 3. Cre protein is…
    • A member of the integrase family (catalyzes an insertion reaction in DNA)
    • Encoded by the E. coli phage P1
    • During cell division, Cre separates dimers of P1 that arise after replication
  • 4. Cre protein is…
    • A member of the integrase family (catalyzes an insertion reaction in DNA)
    • Encoded by the E. coli phage P1
    • During cell division, Cre separates dimers of P1 that arise after replication
  • 5. Cre protein is…
    • A member of the integrase family (catalyzes an insertion reaction in DNA)
    • Encoded by the E. coli phage P1
    • During cell division, Cre separates dimers of P1 that arise after replication
    Replication
  • 6. Cre protein is…
    • A member of the integrase family (catalyzes an insertion reaction in DNA)
    • Encoded by the E. coli phage P1
    • During cell division, Cre separates dimers of P1 that arise after replication
    Homologous Recombination Replication
  • 7. Cre protein is…
    • A member of the integrase family (catalyzes an insertion reaction in DNA)
    • Encoded by the E. coli phage P1
    • During cell division, Cre separates dimers of P1 that arise after replication
    Cre-mediated recombination Homologous Recombination Replication Modified from http://www.esb.utexas.edu/jayaramlab/recomb/Cre.html
  • 8. How does Cre work?
    • Site of action is the loxP site.
    • loxP consists of two 13 bp inverted binding sites separated by a 6 bp spacer .
    • Cleavage sites
    loxP site
  • 9. How does Cre work?
    • 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.
  • 10. How does Cre work?
    • Model of Recombination:
    Strand swapping occurs and the Holliday junction intermediate is formed.
  • 11. How does Cre work?
    • Model of Recombination:
    The proteins undergo an allosteric conformational change. Now, the blue monomers are active for cleavage and the green ones are supressed.
  • 12. How does Cre work?
    • Model of Recombination:
    Modified from http://www.esb.utexas.edu/jayaramlab/model.html
  • 13. Study Objective
    • In floxed (fg2) mice the addition of the protein pops out the gene coding for the function of axon 8 in the  -2 subunit.
    • Can be used selectively to knock out this gene in different regions of the brain
    http://web1.nsac.ns.ca/news/ac_post/1997/aug97/mouse. htm
  • 14. Why knock out  -2 this way? Current Method: Cross-breeding New Method: Protein Injection EMX-1
  • 15. Materials and Methods
    • Expression
      • Growing the E. coli
      • Induce over expression with IPTG
      • Freezing the cells
    • Purification
      • Sonication & centrifugation
      • Ni-NTA resin & elution
    • Preparation
      • Filter
      • Dialysis
        • With aCSF
      • Quantitation
        • Bradford Assay
  • 16. Materials and Methods
    • Expression
      • Growing the E. coli
      • Induce over expression with IPTG
      • Freezing the cells
    • Purification
      • Sonication & centrifugation
      • Ni-NTA resin & elution
    • Preparation
      • Filter
      • Dialysis
        • With aCSF
      • Quantitation
        • Bradford Assay
  • 17. Precipitate?!
    • That’s not supposed to be there!
    Protein crashed out
    • Why?
    • High [salt]
    • pH
    • How do we find out what’s wrong?
    • Recalculated protocol amounts (esp. hydrated salts)
    • pH individual components of aCSF
    • Test every 2 components (of the 3) against each other to see if ppt. forms
  • 18. Results of 2-Component Test aCSF alone = CLEAR aCSF + MgCL2 = CLEAR aCSF + CaCl2 = WHITE PPT.       Is there another protocol for aCSF that might work better?
  • 19. Artificial Cerebrospinal Fluid Formulations
    • NaCl (124 mM)
    • KCl (1.6 mM)
    • Glucose
    • CaCl 2 (2.5 mM)
    • MgCl 2 (1.5 mM)
    • NaHCO 3 (24 mM)
    • KH 2 PO 4 (1.2 mM)
    • Ascorbic Acid (2 mM)
    • NaCl (147 mM)
    • KCl (2.9 mM)
    • Dextrose
    • CaCl 2 (1.7 mM)
    • MgCl 2 (1.6 mM)
    First, we tried: Then, we tried:
  • 20. Artificial Cerebrospinal Fluid Formulations
    • NaCl ( 124 mM )
    • KCl ( 1.6 mM )
    • Glucose
    • CaCl 2 ( 2.5 mM )
    • MgCl 2 ( 1.5 mM )
    • NaHCO 3 (24 mM)
    • KH 2 PO 4 (1.2 mM)
    • Ascorbic Acid (2 mM)
    • NaCl ( 147 mM )
    • KCl ( 2.9 mM )
    • Dextrose
    • CaCl 2 ( 1.7 mM )
    • MgCl 2 ( 1.6 mM )
    First, we tried: Then, we tried:
  • 21. Artificial Cerebrospinal Fluid Formulations
    • NaCl ( 124 mM )
    • KCl ( 1.6 mM )
    • Glucose
    • CaCl 2 ( 2.5 mM )
    • MgCl 2 ( 1.5 mM )
    • NaHCO 3 (24 mM)
    • KH 2 PO 4 (1.2 mM)
    • Ascorbic Acid (2 mM)
    • NaCl ( 147 mM )
    • KCl ( 2.9 mM )
    • Dextrose
    • CaCl 2 ( 1.7 mM )
    • MgCl 2 ( 1.6 mM )
    First, we tried: Then, we tried:
  • 22. Artificial Cerebrospinal Fluid Formulations
    • NaCl ( 124 mM )
    • KCl ( 1.6 mM )
    • Glucose
    • CaCl 2 ( 2.5 mM )
    • MgCl 2 ( 1.5 mM )
    • NaHCO 3 (24 mM)
    • KH 2 PO 4 (1.2 mM)
    • Ascorbic Acid (2 mM)
    • NaCl ( 147 mM )
    • KCl ( 2.9 mM )
    • Dextrose
    • CaCl 2 ( 1.7 mM )
    • MgCl 2 ( 1.6 mM )
    First, we tried: Then, we tried:
  • 23. Artificial Cerebrospinal Fluid Formulations
    • NaCl (124 mM)
    • KCl (1.6 mM)
    • Glucose
    • CaCl 2 (2.5 mM)
    • MgCl 2 (1.5 mM)
    • NaHCO 3 (24 mM)
    • KH 2 PO 4 (1.2 mM)
    • Ascorbic Acid (2 mM)
    • NaCl ( 147 mM )
    • KCl ( 2.9 mM )
    • Dextrose
    • CaCl 2 ( 1.7 mM )
    • MgCl 2 ( 1.6 mM )
    First, we tried: Then, we tried:
  • 24. How was this conclusion reached?
    • Had the same problem making anaerobic media
    • The NaHCO 3 was identified as the ‘problem ingredient’
    • Also yielded white precipitate
    • New buffer system devised (using KH 2 PO 4 and K 2 HPO 4 instead of NaHCO 3 )
  • 25. Other Problems Encountered
    • Shaker temperature
      • Changed from 37 o C to 25 o C.
    • Centrifuge noise
      • Tubes not swinging freely
      • Slight imbalance
  • 26. Results
  • 27. Results
    • Band A : 75.148x -1.1081 = 1.3  x = 38.9 kDa
    • Band B : 75.148x -1.1081 = 2.3  x = 23.25 kDa
    • Band C : 75.148x -1.1081 = 2.7  x = 20.12 kDa
    Desired Protein Product Possible Protein Degradation Products  A   B   C  Ladder Pst-son Pst-B2 Pst-B3 Flo-Th Final 1.3  2.3  2.7 
  • 28. Future Directions
    • Most protein loss occurred early in protocol (before first aliquot)
    • Try it again with the following changes:
      • Less sonication (extra bands are probably degradation products of desired protein)
      • Monitor sonication more closely (checking viscosity)
    • Leave purification steps unchanged (no significant contamination in aliquots)
  • 29. Acknowledgements
    • Clint Earnheart
    • Everyone else in the L ü scher lab
    http://www.criver.com/xruk/transgenics.html - I’m chimeric but I’m cute!