purification of ccs, copper chaperone for superoxide dismutase sean mcintyre lab: dr. joe beckman
TRANSCRIPT
Purification of CCS, copper chaperone for Superoxide
Dismutase
Sean McIntyre
Lab: Dr. Joe Beckman
ALS (Lou Gehrig’s Disease)
• Progressive, neurodegenerative
• 5000 diagnoses per year
• Mutant SOD (metal deficient)
Superoxide Dismutase
O2•-
ONOO-
SOD
Superoxide Dismutase
O2•-
ONOO-
Cellular Proteins (structural, enzymatic)
Nitrated Proteins
- One motor neuron connects to one muscle fiber
Catalytic SOD
CCS
• Assembly of superoxide dismutase- Copper chaperone binds to SOD, inserts copper
• Size- 28.5 kD
one-metal SOD
Why look at CCS?
• SOD mutants of interest are metal deficient mutants, look at mutant SOD-CCS interaction– Different interactions– Why are mutants metal deficient?– Addition/removal of metal from SOD as a means
of “deactivating” mutants
CCS Purification
- Optimizing plasmid and induction temperature- Isolation
- Extraction of protein from bacteria- Anion exchange column- Size separation column
- Preventing aggregation- Copper/Zinc addition- BME addition
Cloning plasmid and Induction temperature
25 kD
37 kD
50 kD
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pET3D18C
pET3D37C
pBAD18C
pBAD37C
zero 3hrs 20hrs zero 3hrs 20hrs zero 3hrs 20hrs control zero 3hrs 20hrs control
Cloning vector and Induction temperature
25 kD
37 kD
50 kD
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pET3D18C
pET3D37C
pBAD18C
pBAD37C
zero 3hrs 20hrs zero 3hrs 20hrs zero 3hrs 20hrs control zero 3hrs 20hrs control
Isolation: First Steps+Cu
Isolation: Anion Exchange Chromatography
Peaks 1 2 3 4 5
Isolation: Anion Exchange Chromatography
25 kD
37 kD
50 kD
25 kD
37 kD
50 kD
pellets peaks1 2 3 4 5
1 2 3 4 5
Isolation: Size exclusion chromatography
• Similar to gel electrophoresis, separates proteins based on size
- Larger size = slower movement
Aggregation
25 kD
37 kD
50 kD
+Cu/Zn -Cu/Zn
Acknowledgements
• Joe Beckman• Nathan Lopez• Kevin Ahern• Casareno, Waggoner, Gitlin (Journal of Biological Chemistry, Sept 11
1998) “The Copper Chaperone CCS Directly Interacts with Copper/Zinc Superoxide Dismutase”
• Estevez, Crow, Sampson, Reiter, Zhuang, Richardson, Tarpey, Barbeito, Beckman (Science, 1999) “Induction of Nitric Oxide – Dependent Apoptosis in Motor Neurons by Zinc-Deficient Superoxide Dismutase”