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WINTERSCHOOL 2010 on Biomolecular Solid-State NMR    January 24 - 29, 2010 – Stowe, Vermont USA    Presentation III: Sampl...
Relative effort devoted to the four pillars.                     80 %               Sample preparation
Expression and purification of membrane proteins I.    Identify target protein.    Select polypeptide sequence.        ...
Codon optimization. DNA 2.0 (www.dna20.com)
Expression and purification II.  Assemble plasmid.  Optimize expression levels in various media.  Optimize expression l...
Expression and purification III: media for isotopic labeling.  Unlabeled samples.      LB  100% uniform 15N.      M9 w...
NP media for selective (by residue type) labeling.                                  Media Comparison Test (OD vs Time)    ...
Selective labeling (by residue type) with NP media:             ~ 3X higher OD and protein yield than M9.    std NP NP M9 ...
Example: p7 of Hepatitis C Virus (HCV).  63 residues.  Two trans-membrane helices.  Oligomers with ion-channel activity...
p7 polypeptide constructs:full-length, C-terminal truncated, and N-terminal truncated.       Codon-optimized sequence of f...
Plasmid.                                           Agarose gel of PCR products for p7 construct insert DNA. The           ...
Expression and purification of p7 polypeptide constructs.
Increased cell density (and yield of protein) using a fermentor.
Solution NMR and solid-state NMR spectra of uniformly 15N                   labeled p7 constructs.Q=0 DHPC micellesq=3.2 D...
Sample preparation in lipids.
Phospholipid bilayer assemblies are characerized by q, the molar      ratio of the long chain lipid to the short chain lip...
NMR of lipid membranes is a well-established technique.    Schindler, H. and Seelig, J. (1975) Deuterium order parameters...
31P   NMR demonstrates that identical phospholipids are affected               differently by different proteins.         ...
31P   NMR spectra consistent with specific modes of interactions             between proteins and phospholipids.          ...
Alignment of protein-containing bicelles is sensitive to lipid              composition and temperature.
Samples with bilayer normals parallel to the field.glass plates     TBBPC bicelles     ‘flipped’ bicelles with lanthanide
Resource for NMR Molecular Imaging of Proteins at UCSD.                                                  Research group"  ...
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Opella l3

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Opella l3

  1. 1. WINTERSCHOOL 2010 on Biomolecular Solid-State NMR January 24 - 29, 2010 – Stowe, Vermont USA Presentation III: Sample preparation. Stanley J. Opella Department of Chemistry and Biochemistry University of California, San Diego La Jolla, California USA
  2. 2. Relative effort devoted to the four pillars. 80 % Sample preparation
  3. 3. Expression and purification of membrane proteins I.  Identify target protein.  Select polypeptide sequence.   Wild-type.   Domain.   Truncation.   Chimera.   Mutations.  Select fusion partner.   KSI.   ΔTrp.   GST.   Bcl-XL.   MBP.  Select cleavage method.   Protease.   Cyanogen bromide.  Obtain codon-optimized gene.
  4. 4. Codon optimization. DNA 2.0 (www.dna20.com)
  5. 5. Expression and purification II.  Assemble plasmid.  Optimize expression levels in various media.  Optimize expression levels in E. coli strains.   BL21 – tightest control of uninduced expression   BL21 (DE3) – high level expression   C41 (DE3) – resistant to membrane protein toxicity  Protein isolation.   Separate/solubilize inclusion bodies.   Nickel column chromatography.  Cleavage.  Protein purification.   HPLC.   FPLC.
  6. 6. Expression and purification III: media for isotopic labeling.  Unlabeled samples.   LB  100% uniform 15N.   M9 with 15N ammonium sulfate.  100% uniform 13C and 15N.   M9 with 13C glucose and 15N ammonium sulfate.   CIL BioExpress.  Tailored 13C labeling.   CIL BioExpress with 15% - 45% uniform 13C and 100% uniform 15N.   Metabolic labeling.   M9 with [2-13C]-glycerol.   M9 with [1,3-13C]-glycerol.   M9 with 2-13C glucose.  Selective (by residue type) 15N and/or 13C amino acids.   M9 with added amino acids.   NP with added amino acids.
  7. 7. NP media for selective (by residue type) labeling. Media Comparison Test (OD vs Time) 14 12 10OD @ 600nm 8 LB 6 M9 NP4XF 4 2 0 0 5 10 15 20 25 Time (hours)
  8. 8. Selective labeling (by residue type) with NP media: ~ 3X higher OD and protein yield than M9. std NP NP M9 M9 two 15N-Leu residuesNP contains salts, amino acids, nucleotides, glucose and other carbon sources
  9. 9. Example: p7 of Hepatitis C Virus (HCV).  63 residues.  Two trans-membrane helices.  Oligomers with ion-channel activity.  Channel blockers have anti-viral activity.  Viroporins.   M2 of influenza.   p7 of BVDV.   Vpu of HIV-1 (not HIV-2 or SIV).   K of Semliki Forest virus.   p10 of avian and Nelson Bay reoviruses.   2B of picornavirus   NS3 of bluetongue virus.   E of MHV (murine hepatitis virus). Lindenbach et al., Nature, 2005
  10. 10. p7 polypeptide constructs:full-length, C-terminal truncated, and N-terminal truncated. Codon-optimized sequence of full-length p7.
  11. 11. Plasmid. Agarose gel of PCR products for p7 construct insert DNA. The 1.5% gel shows the amplification of the sequences for (A) p7,The DNA and protein sequence of the (B) p7TM1 and (C) p7TM2. Lane 1 for each of the gels is thefull-length protein are shown above the base pair size marker 100 Ladder (New England Biolabs).vector map. The pHLV plasmidcontaining the His tagged TrpDLE fusionpartner was used by inserting the p7DNA sequence into the vector betweenthe HinDIII and BamHI restriction sites.The DNA insert included an N-terminalmethionine for cleavage and two C-terminal stop codons.
  12. 12. Expression and purification of p7 polypeptide constructs.
  13. 13. Increased cell density (and yield of protein) using a fermentor.
  14. 14. Solution NMR and solid-state NMR spectra of uniformly 15N labeled p7 constructs.Q=0 DHPC micellesq=3.2 DMPC:DHPC bicelles
  15. 15. Sample preparation in lipids.
  16. 16. Phospholipid bilayer assemblies are characerized by q, the molar ratio of the long chain lipid to the short chain lipid.  Micelles   q=0   T = 50oC – 65oC   Isotropic   Weakly aligned  Isotropic bicelles   q < 1.0 Bo   T = 50oC   Isotropic   Weakly aligned  Magnetically aligned bilayers   q = 2.5 – 10   T = 15oC – 65oC   Completely aligned   Parallel or perpendicular  Unoriented bilayers.
  17. 17. NMR of lipid membranes is a well-established technique.  Schindler, H. and Seelig, J. (1975) Deuterium order parameters in relation to thermodynamic properties of a phospholiped bilayer. A statistical mechanical interpretation. Biochemistry14, 2283–2287.  Davis, J. H., Jeffrey, K. R., Bloom, M., Valic, M. I., and Higgs, T. P. (1976) Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem. Phys. Lett.42, 390–394.  Griffin, R. G. (1976) Observation of the effect of water on the 31P nuclear magnetic resonance spectra of dipalmitoyllecithin. J. Am .Chem. Soc. 98, 851–853.  Kohler, S. J. and Klein, M. P. (1976) 31P nuclear magnetic resonance chemical shielding tensors of phosphorylethanolamine, lecithin, and related compounds:Applications to head-group motion in model membranes. Biochemistry15, 967–974.  Seelig, J. (1977) Deuterium magnetic resonance: theory and application to lipid membranes. Q. Rev. Biophys. 10, 353–418.  Seelig,J. (1978) 31 P nuclear magnetic resonance and the head group structure of phospholipids in membranes. Biochim. Biophys. Acta515, 105–140.  Cullis,P. R. and de Kruijff,B. (1978) The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31 P NMR study. Biochim. Biophys. Acta513, 31–42.  Arnold, K., Gawrisch, K., and Volke, F. (1979) 31 P NMR investigations of phospholipids I. Dipolar interactions and the 31 P NMR lineshape of oriented phospholipid/water dispersions. Stud. Biophys. 75, 189–197.  Davis, J. H. (1983) The description of membrane lipid conformation, order and dynamics by 2 H-NMR. Biochim. Biophys. Acta737, 117–171.  Macdonald, P. M. and Seelig, J. (1988) Anion binding to neutral and positively charged lipid membranes. Biochemistry27, 6769–6775.  Soubias,O.,Saurel,O.,Reat,V.,and Milon,A. (2002) High resolution 13C NMR spectra on ori- ented lipid bilayers: from quantifying the various sources of line broadening to performing 2D experiments with 0.2-0.3 ppm resolution in the carbon dimension. J. Biomol. NMR 24, 15–30.
  18. 18. 31P NMR demonstrates that identical phospholipids are affected differently by different proteins. Unperturbed bilayers Micelle coexistence Fully isotropic Multiple aligned phases Partial bilayer disruption
  19. 19. 31P NMR spectra consistent with specific modes of interactions between proteins and phospholipids. Add protein + + Add protein
  20. 20. Alignment of protein-containing bicelles is sensitive to lipid composition and temperature.
  21. 21. Samples with bilayer normals parallel to the field.glass plates TBBPC bicelles ‘flipped’ bicelles with lanthanide
  22. 22. Resource for NMR Molecular Imaging of Proteins at UCSD. Research group" Lauren Albrecht (GPCR)" Eugene Lin (triple-resonance NMR)" Hua Zhang (Vpu)" Sang Ho Park" Gabriel Cook" Albert Wu" Leah Cho" Christopher Grant " Woo Sung Son" Yan Wang" George Lu" Aubrey Davis" Supported by the National Insitutes of Health" Yanwen Mai" Megan Chu"

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