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

2. Affinity Chromatography Molecule of InterestLigandMatrix Protein Phosphatase-1 Sepharose Microcystin-LR

3. Protein Phosphatase-1 (PP1) Protein Phosphatase-1 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. C-terminal groove Hydrophobic groove Acidic Groove RVXF Motif Binding Site Microcystin-LR Bound to PP1 Binds to active site of PP1 RVXF motif binding site exposed 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. 0 50 100 150 0.300 0.200 0.100 0.000 Microcystin-LR Standard 121.04min Absorbance (206nm) Retention Time (min) HPLC: Buffer A: 0.1%TFA/H 2 O Buffer B: 0.1%TFA/Acetonitrile Rate: 0.3% B/min over 3 hours

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

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

11. Step 2: Add Linker to MCLR

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

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

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

15. 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 Binding Experiments with PP1 repeat Add PP1 Note: Tris-Sepharose resin was used as the “Control “ and ran in parallel with Micrystin-Sepharose resin controlMicrcystin- Sepharose

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

17. 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) Binding Experiments with PP1 Wash And remove supernatant Add PP1 controlMicrcystin- Sepharosel Elute

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

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

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