Updates on Prion Protein Purification Donovan Duggan Bow Suriyamongkol Supervisor: Dr. David Wishart Prion Group Meeting 2 February, 2007.

Updates on Prion Protein Purification

Donovan DugganBow Suriyamongkol

Supervisor: Dr. David Wishart

Prion Group Meeting2 February, 2007

Donor

Purification continues

Optimisation of purification

Steps to remove contaminating upper and lower bands

•Gradient Elution

•Possible double his tag, combined with a gradient elution

Post purification protocols

1) Determine protein concentration

2) Determine optimal buffer for highly concentrated samples

3) Determine protein structure during each step of post purification protocols

1) Determine protein concentration

• All quantification methods are rough approximations.

Absorbance at 280

Hartree-Lowry and modified Lowry Protein assays.

Bicinchoninic Acid (BCA)

Bradford

Kjeldahl

Very rough approximations with visualization in gels or on filter papers.

2) Determination of optimal buffers

• Heavy empirical screen.

Temperature

pH

Ionic strength

Detergents

Glycerol content

Small stabilizing molecules

3) Determine protein configuration before and after any condition

change.

• Circular Dichroism

• Microscopy

W/T ( 2 mg/mL) Day 1 Day 2 Day 1 Day 2

4C 37C 4C 37C

ddH2O O O Hydrophobic reaction

25 mM NH4HCO3 pH 8.0 O O (20mM Tris, 100mM

50 mM NH4HCO3 pH 8.0 O O NaCl, pH 8.0 base buffer)

100 mM NH4HCO3 pH 8.0 O O Glycerol 5% (v/v) O O

Glycerol 10% (v/v) O O

Disulfide bridges Glycerol 15% (v/v) O O

(20mM Tris, 100mM Glycerol 20% (v/v) O O

NaCl, pH 8.0 base buffer)

DTT 5mM O O Ionic reaction

DTT 10mM O O (20mM Tris, pH 8.0

DTT 15mM O O base buffer)

DTT 20mM O O NaCl 100 mM X

NaCl 200 mM O O

NaCl 500 mM O O

KCl 100mM O O

KCl 200mM O O

KCl 500mM O O

Na2PO4 100mM O O

Na2PO4 200mM O O

Na2PO4 500mM N/A N/A

Wild Type (3 mg/mL)

198 ( 4 mg/mL) Day1 198 (2 mg/mL) Day1 Day2

4C 4C 3h RT

Disulfide bridges Disulfide bridges

(20mM Tris, 100mM (20mM Tris, 100mM

NaCl, pH 8.0 base buffer) NaCl, pH 8.0 base buffer)

DTT 5mM X DTT 5mM O O

DTT 10mM X DTT 10mM O O

DTT 15mM X DTT 15mM O O

DTT 20mM X DTT 20mM O O

Hydrophobic reaction Hydrophobic reaction

(20mM Tris, 100mM (20mM Tris, 100mM

NaCl, pH 8.0 base buffer) NaCl, pH 8.0 base buffer)

Glycerol 5% (v/v) X Glycerol 5% (v/v) O O

Glycerol 10% (v/v) X Glycerol 10% (v/v) O O

Glycerol 15% (v/v) X Glycerol 15% (v/v) O O

Glycerol 20% (v/v) X Glycerol 20% (v/v) O O

Ionic reaction Ionic reaction

(20mM Tris, pH 8.0 (20mM Tris, pH 8.0

base buffer) base buffer)

NaCl 100 mM X NaCl 100 mM O O

NaCl 200 mM X NaCl 200 mM O O

NaCl 500 mM X NaCl 500 mM O O

KCl 100mM X KCl 100mM O O

KCl 200mM X KCl 200mM O O

KCl 500mM X KCl 500mM O O

Na2PO4 100mM X Na2PO4 100mM O O

Na2PO4 200mM X Na2PO4 200mM O O

Na2PO4 500mM X Na2PO4 500mM O O

F198S (4 mg/mL and 2 mg/mL)

178 ( 4 mg/mL) Day 1 Day 2 Day 3

4C RT 37C

10 mM K2PO4

pH 6.0 X

pH 6.6 X

pH 7.2 X

50 mM K2PO4

pH 6.0 X

pH 6.6 X

pH 7.2 X

100 mM K2PO4

pH 6.0 X

pH 6.6 X

pH 7.2 X

10 mM NaOAc

pH 4.0 O O O

pH 4.5 O O O

pH 5.0 O O O

pH 5.5 X

25 mM NaOAc

pH 4.0 O O O

pH 4.5 O O O

pH 5.0 O O O

pH 5.5 X

50 mM NaOAc

pH 4.0 O O O

pH 4.5 O O O

pH 5.0 O O O

pH 5.5 X

D178N (4 mg/mL)

200 ( 2 mg/mL) Day 1 Day 2 Day 3

4C RT 37C

10 mM K2PO4

pH 6.0 O O X

pH 6.6 O O X

pH 7.2 O O X

50 mM K2PO4

pH 6.0 O O X

pH 6.6 O O X

pH 7.2 O O X

100 mM K2PO4

pH 6.0 O O X

pH 6.6 O O X

pH 7.2 O O X

10 mM NaOAc

pH 4.0 O O O

pH 4.5 O O O

pH 5.0 O O O

pH 5.5 O O O

25 mM NaOAc

pH 4.0 O O O

pH 4.5 O O O

pH 5.0 O O O

pH 5.5 O O O

50 mM NaOAc

pH 4.0 O O O

pH 4.5 O O O

pH 5.0 O O O

pH 5.5 O O O

E200K (2 mg/mL)

Summary of Solubility of The Proteins

Wild type (2 mg/mL) , pI 8.86 almost all the buffers tested, including ddH2O

F198S (2 mg/mL), pI 8.86 20 mM Tris, 100 mM NaCl, pH 8.0

D178N (4 mg/mL), 9.08 10 to 50 mM NaOAc, pH 4.0-5.0

E200K (2 mg/mL), 9.23 10 to 50 mM NaOAc, pH 4.0-5.5

Donovan’s results for P102L mutant

Sodium acetate buffer (up to 3 days at 37C)- 10 to 100 mM, pH 4.0- 25 to 100 mM, pH 4.5- 50 to 100 mM, pH 5.0- 10 to 50 mM, pH 5.5

P102L ( ? mg/mL) is stable in:

Potassium phosphate buffer (up to 1 day at 37C)- 10 to 100 mM, pH 8.0

SDS-PAGE Gels of Purified Proteins

Wild type

15

20

kDa

Total amount of 45 milligrams per 1L of culture

F198S

1520

kDa

Total amount of 35 milligrams per 1L of culture

Bradford Std Curve

y = 0.8084x + 0.018

R2 = 0.9977

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.2 0.4 0.6 0.8 1

BSA (mg/mL)

AB

S 5

95 n

m

Protein Quantification

Acknowledgements

Dr. David WishartValentyna Semenchenko

Thank you.