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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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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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

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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