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

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

7. Synthesis of fusion protein Fusion protein Cell extract

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

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

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

11. Pro-domain proR58 stripped from column with 0.1M H 3 PO 4 pH 2.1

12. Purified proteins Streptococcal protein G B domain 56 aa Streptococcal protein G a domain 45 aa Protein G B mutant G31156 aa Staphylococcal Protein A B domain 56 aa Protein A B mutant A21956 aa E. coli hypothetical Yab117 aa Bovine  subunit of transducin350 aa M. thermautotrophicus CDC6379 aa A. victoria Green Fluorescent Protein238 aa

13. Conformational switching A219 G311

14. 15 N HSQC spectra G311 25˚C 2˚C

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

16.  -subunit bovine transducin (350aa)

17. Processing activity: K d 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. K d of processed proR58 and SBT is < 0.1nM Non-specific cleavage is undetectable

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

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

20. 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