Laura TZEPLAEFF

07-31-2016 to 10 -21-2016

BITAN’S LAB

SUPERVISOR : R av inder MALIK, PhD

SOD1 PURIFICATION

2

Introduction

Amyotrophic lateral sclerosis (ALS): Describe by Charcot, 1869.

Image from: https://fr.wikipedia.org/wiki/Jean-Martin_Charcot

Jean Martin Charcot

- Charcot disease, Lou Gehrig’s disease,…

- fatal human neurodegenerative disease within 1–5 years of onset

- affecting the motor neurons

- prevalence of 2–3 per 100,000 people

- 90-95% sporadic form (sALS) and 5-10% familial form (fALS)

3

Introduction

Neuropathology:

- progressive muscle weakness, atrophy and spasticity

- degeneration and death of upper or lower motor neurons respectively in the brain and spinal cord

Image modified from: http://www.frisksomenfisk.com

Muscle

AxonAxon

Atrophiedmuscle

Cell body

Normal nerve cellAnd muscle

ALS-affected nerve Cell and muscle

4

Introduction

Cu/Zn-superoxide dismutase (SOD1):

- (≈150) dominant mutations, 20% fALS, causing SOD1 misfolding

- abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons

- Causing death of motor neurons

Image modified from:

5

Introduction

Cu/Zn-superoxide dismutase (SOD1):

- antioxidant enzyme detoxifying superoxide radicals

- native form is a 32 kDa homodimer

- enzymatically active by binding copper and zinc ions and forming highly conserved intramolecular disulfide bonds

SOD1 reaction:2O2

.- → H2O2 + O2

Metal loss

Structural transition

ALS

Image modified from: http://www.pnas.org

6

Overview

Yeast Cell Culture

Cell Lysis

Ammonium Sulfate Precipitation

Hydrophobic Interaction Chromatography

Ion Exchange Chromatography

Size Exclusion Chromatography

Protein Purified

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Overview

Yeast Cell Culture Cell lyses Ammonium sulfate precipitation Hydrophobic interaction

chromatography Enzyme activity assay SDS PAGE Dialysis

Anionic exchange chromatography Protein concentration Size exclusion chromatography BCA assay Flash freeze

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Yeasts Cell Culture

Inoculation:

Cell type: EG118 Δ SOD1 yeast colony (lacking cytosolic

SOD1)

Plasmid:

- YEp 351 : Yeast/E-coli plasmid vector

- hWT/hALS gene

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Yeasts Cell Culture

Yeast Peptone and Dextrose Growth media, pH= 7.0:

Bacto Peptone 20 g/L Bacto Yeast Extract 10 g/L Copper chloride

23.33 g/L Glucose 20 g/L

Synthetic Defined –Leucine Selective Growth Media, pH=7.0:

Yeast Nitrogen Base 30 g/L Ammonium Sulfate 83.33 g/L Monosodium Phosphate

23.33 g/L Amino Acid Mix (-Leu) 26.83 g/L Glucose 20 g/L (Bacto Agar, for solid media) 0.3 g/L

Control EG118YEp 351

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Cell lyses

Cell Lysis Buffer, pH = 7.0:

Tris 250 mM

NaCl 150 mM

EDTA 0.1 mM

l

2 min blending

2 min ice

0.5 mm Glass beads:

300 mL each 500 mL

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Ammonium sulfate precipitation (salting out)

Reagents:

Ammonium Sulfate2.4 M

Pulverized

High salt concentration: Salting out

- Water interact with salt- Protein–water binding disruption- Protein insoluble- Hydrophobic interactions,

aggregation of high MW.Image from : http://nptel.ac.in/courses

2

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Hydrophobic Interaction Chromatography (HIC)

Low salt concentration

High salt concentration

Image modified from : Tao Chen, 2015

HIC buffer, pH= :

Sodium Phosphate Monobasic 1 M NaCl

3 M EDTA

0.5 M Support matrix

Hydrophobic ligand Phenyl Sepharose 6

Hydrophobic portion

Hydrophilic portion

Salt : Ammonium Sulfate

Low salt concentration:

- Water shielding reforming - Protein soluble again - Proteins detached from

hydrophobic matrix- Low hydrophobic to high

hydrophobic proteins elution (90 fractions, 10 mL)

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Hydrophobic Interaction Chromatography (HIC)

Results: Enzyme activity assay & SDS-PAGE

Enzyme activity essay:- Hypoxanthine: Purine derivate- Xanthine Oxidase: Catalyze the oxidation of

hypoxanthine to uric acid & generate reactive oxygen species (ROS, ex: O2

.-)- XTT: Reduced to colored orange derivative named

Formazan with O2.-.

- Catalase: Catalyzes the decomposition of hydrogen peroxide to water and oxygen (2 H2O2 → 2H2O + O2).

Image 2 from: Christian Corrales, UCLA

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Hydrophobic Interaction Chromatography (HIC)

1- Mark 122- Fraction 113- Fraction 124- Fraction 135- Fraction 146- Fraction 167- Fraction 188- Fraction 209- Fraction 2210- Fraction 2311- Fraction 2412- Fraction 2513- Flow through14- Wash before HIC15- WT SOD1

3.56.014.4

21.531.036.5

55.466.397.4

200116.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

kDa

Results: Enzyme activity assay & SDS-PAGE

Reducing SDS-PAGE : SOD1 E21K after

15

Dialysis

Modified from : http://www.siumed.edu

Spectra/Por molecularporous membrane tubing of

MWCO 6-8 kDa

Potassium Phosphate2.5 mM, pH=7.0

Sodium Phosphate Monobasic 1 M

At start ofthe dialysis

a)

At equilibriumb)

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Anionic Exchange Chromatography

Image modified from : Elena Komkova, 2010

Mat

rix

Mat

rix

High salt concentration

Low salt concentration

High salt concentration, pH=7.0:

Potassium Phosphate 200 mM

Shielding of the protein, Low charged to high charged proteins elution(60 fractions, 10 mL)

Low salt concentration, pH=7.0:

Potassium Phosphate 2.5 mM

Negative proteins bind to the positive matrix (diethylaminoethanol Sepharose)

Matrix : Diethylaminoethyl Cellulose

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1- Mark 122- Fraction 103- Fraction 114- Fraction 165- Fraction 216- Fraction 267- Fraction 318- Fraction 369- Fraction 4110- Fraction 4611- HIC 112- Flow through13- Wash 1 before DEAE14- Wash 2 after DEAE15- WT SOD1

3.56.0

14.421.531.036.5

55.466.397.4

200116.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

kDa

Anionic Exchange Chromatography

Results: Enzyme activity assay & SDS-PAGE

Reducing SDS-PAGE : SOD1 E21K after DEAE

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Protein concentration

Image modified from : https://www.thermofisher.com

Ultrafiltration Discs, filter diameter

76mm and 44.5mm Molecule

s ≤ 10 kDa

L

Millipore Ultracel 10

kDa

SOD 1

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Size Exclusion Chromatography

Image modified from : www.biochimiedesproteines

Reagents:

Potassium Phosphate20 mM

Sodium Chloride 80 mM

Elution: 90 fractions, 5 mL

- High MW: Avoid the pores and beads

(Superdex 75)

- Medium MW: slowed down by pores

- Low MW: Flow inside poresl Hig

h M

W lLo

w M

W

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Fractions

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Size Exclusion Chromatography

kDa

Results: Enzyme activity assay & SDS-PAGE

Reducing SDS-PAGE :E21K-SOD1 after G-751- Mark 122- Fraction 153- Fraction 164- Fraction 175- Fraction 186- Fraction 197- Fraction 208- Fraction 219- Fraction 2210- Fraction 2311- Fraction 2412- Load13- Flow through G75♯214- Wash after G75♯215- WT SOD13.5

6.0

14.421.531.036.5

55.466.397.4

200116.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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BCA assay

Standard bovine serum albumin concentration :

µg proteins/µL

0.125 0.50 1.00 1.5

0.00 0.25 0.75 1.25??

µg SOD1/µL

SOD1 protein solution:

30 minutes, 37˚C

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Remove Cu/Zn (Dialysis) & Flash Freeze

3x 4L Buffer 1, pH= 3.8:

EDTA 10 mM

Sodium Acetate 100 mM

Glacial Acetic Acid Until pH=3.8

3x 4L Buffer 2, pH= 3.8:

NaCl 100 mM

Sodium Acetate 100 mM

Glacial Acetic Acid Until pH=3.8 3x 4L Buffer 3, pH = 7.0:

Potassium Phosphate 10 mM

Aliquot, flash freeze in liquid nitrogen

Storage in -80 freeze

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Thank You !