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Affinity Chromatography: Homemade Microcystin-Sepharose Column

Affinity Chromatography: Homemade Microcystin-Sepharose Column. Cindy Lee May 1, 2006. Sepharose. Protein Phosphatase-1. Microcystin-LR. Affinity Chromatography. Molecule of Interest. Ligand. Matrix. Protein Phosphatase-1. Protein Phosphatase-1 (PP1).

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Affinity Chromatography: Homemade Microcystin-Sepharose Column

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  1. Affinity Chromatography:HomemadeMicrocystin-Sepharose Column Cindy Lee May 1, 2006

  2. Sepharose Protein Phosphatase-1 Microcystin-LR Affinity Chromatography Molecule of Interest Ligand Matrix

  3. Protein Phosphatase-1 Protein Phosphatase-1 (PP1) • Part of the Ser/Thr Phosphatase Family • Important enzyme in the regulation of many cellular pathways • Involved in reversible phosphorylation of proteins • Must counterbalance the activity of several different protein kinases • Tightly regulated by regulatory subunits

  4. What is Microcystin? • Cyclic heptapeptide • Hepatotoxin found in blue-green algae (cyanobacteria) • Potent inhibitor of protein phosphatase-1 (PP1) • Immediate binding • Covalent binding to Cys-273 of PP1 • >50 kinds • MC-LR and MC-LL are the most common

  5. Microcystin-LR Bound to PP1 Acidic Groove • Binds to active site of PP1 • RVXF motif binding site exposed C-terminal groove RVXF Motif Binding Site Hydrophobic groove Goldberg et al. Nature 376 745-735 (1995)

  6. Uses of Microcystin-Sepharose Affinity Chromatography • Purify PP1 • Bind regulatory proteins of PP1 to PP1 that is bound to the column • RVXF motif binding site is exposed • Purify PP2A • Part of the same Protein Ser/Thr phosphatase family as PP1 • Also inhibited by microcystin • In the literature, there were problems with eluting the PP2A

  7. How to Create a Microcystin-Sepharose Column • Step 1: Obtain Microcystin-LR (MCLR) • Step 2: Add linker to MCLR MCLR + • Step 3: React MCLR with the linker to N-hydroxysuccinimide (NHS) activated-Sepharose + Moorhead et. al. FEBS Letters 356 46-50 (1994)

  8. Microcystin-LR Standard HPLC: Buffer A: 0.1%TFA/H2O Buffer B: 0.1%TFA/Acetonitrile Rate: 0.3% B/min over 3 hours 0.300 0.200 0.100 0.000 121.04min Absorbance (206nm) 0 50 100 150 Retention Time (min)

  9. Step 1: Obtain Microcystin-LR -fractions were collected and pooled from an HPLC purification of microcystin from cyanobacteria from Little Beaver Lake in 1992 0.300 0.200 0.100 0.000 121.27 min Absorbance (206nm) 0 50 100 150 Retention Time (min)

  10. Comparison: Standard vs Pooled Fractions 0.200 0.100 0.000 0.100 0.000 121.04 min Standard Absorbance (206nm) 121.27 min Pooled Fractions 0 50 100 150 Retention Time (min)

  11. Step 2: Add Linker to MCLR

  12. Comparison: Before vs After Reaction with Linker 0.200 0.100 0.000 0.400 0.200 0.000 121.27 min Microcystin pool Absorbance (206nm) 113.89;115.02 min After Reaction with Linker 0 50 100 150 Retention Time (min)

  13. Step 3: React MCLR with Linker to NHS-activated Sepharose

  14. Comparison: Before vs After Reaction with Sepharose 0.400 0.200 0.000 0.100 0.000 113.89;115.02 min After reaction with linker Absorbance (206nm) Supernatant after reaction with NHS-activated Sepharose 0 50 100 150 Retention Time (min)

  15. Binding Experiments with PP1 • Determine the Binding Capacity of the Microcystin-Sepharose • Add increments of PP1 to the resin • Supernatant was tested for activity and used to determine the amount of PP1 that bound Add PP1 repeat control Micrcystin-Sepharose Note: Tris-Sepharose resin was used as the “Control “ and ran in parallel with Micrystin-Sepharose resin

  16. Total PP1 Bound = 14.4+15.2+16.1+13.3 = 59g 50L of resin contains ~14.3g of MCLR: How much PP1 can 1mg of MCLR bind? 59gPP1 * 1mgMCLR / 14.3gMCLR = 4.2mgPP1

  17. Binding Experiments with PP1 • Determine the Binding Capacity of the Microcystin-Sepharose • Using Microcystin-Sepharose for purifying PP1 • 1) Bind PP1 to resin • 2) Wash resin (0.3M NaCl) • 3) Elute PP1 (3M NaSCN) Add PP1 Wash And remove supernatant Elute control Micrcystin-Sepharosel

  18. PP1 Purification Experiment MC-Seph Resin MC-Seph Resin Ctrl Resin MC-Seph Ctrl Resin PP1 For binding Ctrl MW markers After incubation with PP1 After Elution with 3M NaSCN Elution (3M NaSCN) (kDa) 75 50 37 25 20 15 10 Ctrl = control:Tris-Sepharose MC-Seph = Microcystin Sepharose

  19. MC-Seph Resin MC-Seph Resin Ctrl Resin MC-Seph Ctrl Resin PP2A For binding Ctrl MW markers Elution (Okadiac Acid) After incubation with PP2A After Elution with Okadaic Acid (kDA) 75 50 37 25 20 15 10 PP2A Purification Experiment

  20. Future Work… • Repeat Experiments with PP2A • Get a more definite result • Try binding regulatory proteins to PP1 that is bound to the column

  21. Acknowledgements • Holmes Lab • Especially Marcia Craig

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