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Purification of Prion Proteins

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.

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Purification of Prion Proteins

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

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

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

  7. 1st elution (refolded) 9 10 11 12 14 15 16 19 20 15 10 Wild Type SHPrPc(90-231) (18 kDa) 1st elution (refolded) 2nd elution (refolded) 3rd elution (unfolded) 2nd elution (refolded) 3rd elution (unfolded) kDa 2nd kDa 11 13 15 20 15 8 10 11 12 13 10 3rd

  8. 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

  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. Acknowledgements Dr. David Wishart Valentyna Semenchenko Thank you.

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