one-step affinity purification and processing of fusion proteins philip bryan
TRANSCRIPT
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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S189 S190 vs pR8 FKAM
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Processing of fusion protein by SBT-M
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Processing of fusion protein by SBT-M
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Processing of fusion protein by SBT-M
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Processing of fusion protein by SBT-S
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Processing of fusion protein by SBT-M
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S189 S190 vs pR8 FKAM
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Processing of fusion protein by SBT-S
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Processing of fusion protein by SBT-S
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Processing of fusion protein by SBT-S
Precise fusion of target protein onto pro-region
Nde I
ProR58
Target protein
Eco R1 Hind III Sal I
Expression Vector pG58
Synthesis of fusion protein
Fusion protein
Cell extract
Binding of fusion protein to Sbt column
Flow-throughfrom column
Loading on column
Elution of processed target protein
Elution after overnight incubation
Processed target protein
Purification of fusion protein
Fusion protein
Acid elution -No incubation
Pro-domain proR58 stripped from column with 0.1M H3PO4 pH 2.1
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 subunit of transducin 350 aa• M. thermautotrophicus CDC6 379 aa• A. victoria Green Fluorescent Protein 238 aa
Conformational switching
A219 G311
15N HSQC spectra
G311
25˚C
2˚C
-subunit bovine transducin (350aa)load Fractions
1 2 3 4 5 6
pooled
-subunit bovine transducin (350aa)
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
Binding conditions:
• pH 5-10
• 0-1M NaCl
• 0-2M urea (folding on the column)
• 0-1M Gu-HCl
Column is tolerant of:
• EDTA
• PMSF
• Protease inhibitor cocktails
• Reducing agents
• 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