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

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

1. Gene Isolation (http://users.ugent.be/~avierstr/principles/pcr.html)

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

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)

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

Wild Type SHPrPc(90-231) (18 kDa) 1st elution (refolded) 2nd elution (refolded) 3rd elution (unfolded) 1st elution (refolded) 10 15 20 9 10 11 12 14 15 16 19 2nd elution (refolded) 3rd elution (unfolded) kDa kDa 2nd 11 13 15 20 15 8 10 11 12 13 10 3rd

SHPrPc(90-231) 198 (Phe  Ser) Mutant (18 kDa) 1st elution (refolded) 2nd elution (unfolded) 2nd elution (unfolded) 1st elution (refolded) 13 14 15 16 17 18

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

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