Affinity Chromatography:Homemade

Microcystin-Sepharose ColumnCindy Lee

May 1, 2006

Affinity Chromatography

Molecule of Interest Ligand Matrix

Protein Phosphatase-1

Sepharose

Microcystin-LR

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

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

C-terminalgroove

Hydrophobicgroove

AcidicGroove

RVXF MotifBinding 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)

Uses of Microcystin-Sepharose Affinity Chromatography Purify PP1 Bind regulatory proteins of PP1 to PP1

that is bound to the columnRVXF motif binding site is exposed

Purify PP2A Part of the same Protein Ser/Thr phosphatase

family as PP1Also inhibited by microcystin In the literature, there were problems with

eluting the PP2A

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)

0 50 100 150

0.300

0.200

0.100

0.000

Microcystin-LR Standard

121.04min

Ab

sorb

ance

(2

06n

m)

Retention Time (min)

HPLC:Buffer A: 0.1%TFA/H2OBuffer B: 0.1%TFA/AcetonitrileRate: 0.3% B/min over 3 hours

0 50 100 150

0.300

0.200

0.100

0.000

121.27 min

Step 1: Obtain Microcystin-LRA

bso

rban

ce (

20

6nm

)

Retention Time (min)

-fractions were collected and pooled from an HPLC purification of microcystin from cyanobacteria from Little Beaver Lake in 1992

121.27 min

121.04 min

Standard

Pooled Fractions

Comparison: Standard vs Pooled Fractions

0 50 100 150Retention Time (min)

0.200

0.100

0.000

0.100

0.000

Ab

sorb

ance

(2

06n

m)

Step 2: Add Linker to MCLR

113.89;115.02 min

121.27 minMicrocystin

pool

After Reaction with Linker

0 50 100 150Retention Time (min)

0.200

0.100

0.000

0.400

0.200

0.000

Ab

sorb

ance

(2

06n

m)

Comparison: Before vs After Reaction with Linker

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

After reaction with linker

Supernatant after reaction

with NHS-activatedSepharose

Comparison: Before vs After Reaction with Sepharose

0 50 100 150

0.400

0.200

0.000

0.100

0.000

Ab

sorb

ance

(2

06n

m)

Retention Time (min)

113.89;115.02 min

Determine the Binding Capacity of the Microcystin-SepharoseAdd increments of PP1 to the resinSupernatant was tested for activity and used to

determine the amount of PP1 that bound

Binding Experiments with PP1

repeatAdd PP1

Note: Tris-Sepharose resin was used as the “Control “ and ran in parallel with Micrystin-Sepharose resin

control Micrcystin-Sepharose

50L of resin contains ~14.3g of MCLR:

How much PP1 can 1mg of MCLR bind?59gPP1 * 1mgMCLR / 14.3gMCLR = 4.2mgPP1

Amount of PP1 Bound After Each Addition of 17.5 1 50g of PP to -L of Microcystin

Sepharose Resin

0

2

4

6

8

10

12

14

16

18

1 2 3 4

#Addition

1 Amount of PP

Unbound

Bound

Total PP1 Bound = 14.4+15.2+16.1+13.3 = 59g

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

WashAnd

removesupernatant

Add PP1

control Micrcystin-Sepharosel

Elute

(kDa)

75

50

37

25

20

15

10

PP1 Purification Experiment

MWmarkers

PP1For

binding

Ctrl Resin

Ctrl Resin

MC-Seph Resin

MC-Seph Resin Ctrl

MC-Seph

After incubation with PP1

After Elution with 3M NaSCN

Elution(3M NaSCN)

Ctrl = control:Tris-SepharoseMC-Seph = Microcystin Sepharose

PP2A Purification Experiment

(kDA)

75

50

37

25

20

15

10

MWmarkers

PP2AFor

binding

Ctrl Resin

Ctrl Resin

MC-Seph Resin

MC-Seph Resin Ctrl

MC-Seph

After incubation with PP2A

After Elution with Okadaic Acid

Elution(Okadiac Acid)

Future Work…

Repeat Experiments with PP2AGet a more definite result

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

Acknowledgements

Holmes LabEspecially Marcia Craig