Onestep 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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(No Transcript)
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(No Transcript)
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Precise fusion of target protein onto pro-region
Target protein
ProR58
Nde I
Eco R1 Hind III Sal I
Expression Vector pG58
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Synthesis of fusion protein
Cell extract
Fusion protein
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Binding of fusion protein to Sbt column
Flow-through from 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
Acid elution - No incubation
Fusion protein
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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 a-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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25C
15N HSQC spectra G311
2C
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a-subunit bovine transducin (350aa)
load
pooled
Fractions 1 2
3 4 5 6
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a-subunit bovine transducin (350aa)
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Processing activity

• Kd of fusion protein and SBT is lt 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 lt 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