1. Supervisor: Dr. David Wishart
Purification of Prion Proteins
Bow Suriyamongkol
Supervisor: Dr. David Wishart
Prion Group Meeting
6 October, 2006

2. Over-expression and Purification of a Recombinant Protein
1. Gene Isolation
2. Gene Construct Preparation
3. Over-expressing the Target Protein in Expression Host
4. Purification of the Target Protein
5. Modification and Characterization of the Target Protein

3. 1. Gene Isolation (

4. 2. Gene Construct Preparation
pET-15b (protein expression vector)
His-tag
T7 promoter
T7term
SHPrPc(90-231)
AmpR
Thrombin
Syrian hamster prion gene in pET-15b vector

5. 3. Over-expressing the Target Protein in Expression Host
E. coli BL21(DE3)
protein expression host
pET-15b
(adapted from pET system manual 11th edition, page 49)

6. 4. Purification of the Target Protein
Native Condition
Denaturing Condition
Cell
lysis
Native lysis buffer
Denaturing lysis buffer
Ni-NTA
resin
wash
elute
Native elution buffer
Native wash buffer
Denaturing elution buffer
Denaturing wash buffer
Pure His-tagged protein
His-tag
binding

7. Wild Type SHPrPc(90-231) (18 kDa)
1st elution (refolded)
2nd elution (refolded)
3rd elution (unfolded)
1st elution (refolded) 10 15 20
2nd elution (refolded) 3rd elution (unfolded)
kDa
kDa
2nd 20 15 10
3rd

8. SHPrPc(90-231) 198 (Phe  Ser) Mutant (18 kDa)
1st elution (refolded)
2nd elution (unfolded)
2nd elution (unfolded)
1st elution (refolded)

9. 5. Modification and Characterization
of the Target Protein
Refolding denatured protein after purification
Thrombin cleavage
Quantify and Concentrating the purified protein sample
Button test by microdialysis

10. Valentyna Semenchenko
Acknowledgements
Dr. David Wishart
Valentyna Semenchenko
Thank you.