One-step affinity purification and processing of fusion proteins
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One-step affinity purification and processing of fusion proteins
Philip Bryan
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One-step purification:
• affinity purification of fusion proteins,
• removal of the fusion domain
• isolation of the target protein
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Target protein is fused onto an engineered pro-region of subtilisin
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Precise fusion of target protein onto pro-region
Nde I
ProR58
Target protein
Eco R1 Hind III Sal I
Expression Vector pG58
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Synthesis of fusion protein
Fusion protein
Cell extract
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Binding of fusion protein to Sbt column
Flow-throughfrom column
Loading on column
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Elution of processed target protein
Elution after overnight incubation
Processed target protein
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Purification of fusion protein
Fusion protein
Acid elution -No incubation
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Pro-domain proR58 stripped from column with 0.1M H3PO4 pH 2.1
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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
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Conformational switching
A219 G311
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15N HSQC spectra
G311
25˚C
2˚C
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-subunit bovine transducin (350aa)load Fractions
1 2 3 4 5 6
pooled
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-subunit bovine transducin (350aa)
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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
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Binding conditions:
• pH 5-10
• 0-1M NaCl
• 0-2M urea (folding on the column)
• 0-1M Gu-HCl
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Column is tolerant of:
• EDTA
• PMSF
• Protease inhibitor cocktails
• Reducing agents
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• 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