Presentation is loading. Please wait.

Presentation is loading. Please wait.

Purification of CCS, copper chaperone for Superoxide Dismutase Sean McIntyre Lab: Dr. Joe Beckman.

Similar presentations


Presentation on theme: "Purification of CCS, copper chaperone for Superoxide Dismutase Sean McIntyre Lab: Dr. Joe Beckman."— Presentation transcript:

1 Purification of CCS, copper chaperone for Superoxide Dismutase Sean McIntyre Lab: Dr. Joe Beckman

2 ALS (Lou Gehrig’s Disease) Progressive, neurodegenerative 5000 diagnoses per year Mutant SOD (metal deficient)

3 Superoxide Dismutase O2-O2- ONOO - SOD

4 Superoxide Dismutase O2-O2- ONOO - Cellular Proteins (structural, enzymatic) Nitrated Proteins - One motor neuron connects to one muscle fiber Catalytic SOD

5 CCS Assembly of superoxide dismutase- Copper chaperone binds to SOD, inserts copper Size- 28.5 kD one-metal SOD

6 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

7 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

8 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 pET3D 18  C pET3D 37  C pBAD 18  C pBAD 37  C zero 3hrs 20hrs zero 3hrs 20hrs zero 3hrs 20hrs control zero 3hrs 20hrs control

9 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 pET3D 18  C pET3D 37  C pBAD 18  C pBAD 37  C zero 3hrs 20hrs zero 3hrs 20hrs zero 3hrs 20hrs control zero 3hrs 20hrs control

10 Isolation: First Steps +Cu

11 Isolation: Anion Exchange Chromatography Peaks 1 2 3 4 5

12 Isolation: Anion Exchange Chromatography 25 kD 37 kD 50 kD 25 kD 37 kD 50 kD pelletspeaks 1 2 3 4 5

13 Isolation: Size exclusion chromatography Similar to gel electrophoresis, separates proteins based on size -Larger size = slower movement

14 Aggregation 25 kD 37 kD 50 kD +Cu/Zn -Cu/Zn

15 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”


Download ppt "Purification of CCS, copper chaperone for Superoxide Dismutase Sean McIntyre Lab: Dr. Joe Beckman."

Similar presentations


Ads by Google