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One-step affinity purification and processing of fusion proteins

One-step affinity purification and processing of fusion proteins. Philip Bryan. One-step purification:. affinity purification of fusion proteins, removal of the fusion domain isolation of the target protein. Target protein is fused onto an engineered pro-region of subtilisin.

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One-step affinity purification and processing of fusion proteins

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  1. One-step affinity purification and processing of fusion proteins Philip Bryan

  2. One-step purification: • affinity purification of fusion proteins, • removal of the fusion domain • isolation of the target protein

  3. Target protein is fused onto an engineered pro-region of subtilisin

  4. Precise fusion of target protein onto pro-region Target protein ProR58 Nde I Eco R1 Hind III Sal I Expression Vector pG58

  5. Synthesis of fusion protein Cell extract Fusion protein

  6. Binding of fusion protein to Sbt column Flow-through from column Loading on column

  7. Elution of processed target protein Elution after overnight incubation Processed target protein

  8. Purification of fusion protein Acid elution - No incubation Fusion protein

  9. Pro-domain proR58 stripped from column with 0.1M H3PO4 pH 2.1

  10. Purified proteins • Streptococcal protein GB domain 56 aa • Streptococcal protein Ga domain 45 aa • Protein GB mutant G311 56 aa • Staphylococcal Protein AB domain 56 aa • Protein AB mutant A219 56 aa • E. coli hypothetical Yab 117 aa • Bovine a-subunit of transducin 350 aa • M. thermautotrophicus CDC6 379 aa • A. victoria Green Fluorescent Protein 238 aa

  11. Conformational switching A219G311

  12. 25˚C 15N HSQC spectra G311 2˚C

  13. a-subunit bovine transducin (350aa) load pooled Fractions 1 2 3 4 5 6

  14. a-subunit bovine transducin (350aa)

  15. Processing activity: • Kd of fusion protein and SBT is < 1nM • Binding of fusion protein to SBT is fast • Processing is a slow, single turn-over reaction • Precise N-terminus of target protein produced. • Kd of processed proR58 and SBT is < 0.1nM • Non-specific cleavage is undetectable

  16. Binding conditions: • pH 5-10 • 0-1M NaCl • 0-2M urea (folding on the column) • 0-1M Gu-HCl

  17. Column is tolerant of: • EDTA • PMSF • Protease inhibitor cocktails • Reducing agents

  18. Biochemistry Patrick Alexander Kathryn Fisher Joel Hoskins Biao Ruan Sergei Ruvinov Susan Strausberg Lan Wang NIH GM42560 X-ray Orna Almog Travis Gallagher Gary Gilliland NMR John Orban Nese Sari

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