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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

Affinity Chromatography:HomemadeMicrocystin-Sepharose Column

Cindy Lee

May 1, 2006


Affinity chromatography

Sepharose

Protein

Phosphatase-1

Microcystin-LR

Affinity Chromatography

Molecule of Interest

Ligand

Matrix


Protein phosphatase 1 pp1

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


What is microcystin
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


Microcystin lr bound to pp1
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)


Uses of microcystin sepharose affinity chromatography
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


How to create a microcystin sepharose column
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)


Microcystin lr standard
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)


Step 1 obtain microcystin lr
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)


Comparison standard vs pooled fractions
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)



Comparison before vs after reaction with linker
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)



Comparison before vs after reaction with sepharose
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)


Binding experiments with pp1
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


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


Binding experiments with pp11
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


Pp1 purification experiment
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


Pp2a purification experiment

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


Future work
Future Work…

  • Repeat Experiments with PP2A

    • Get a more definite result

  • Try binding regulatory proteins to PP1 that is bound to the column


Acknowledgements
Acknowledgements

  • Holmes Lab

    • Especially Marcia Craig


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