Transcript One-step affinity purification and processing of fusion proteins Philip Bryan

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
1
S189 S190 vs pR8 FKAM
Processing
fusion
protein
by
SBT-M
Processing
of
fusion
protein
byby
SBT-M
Processingof
of
fusion
protein
by
SBT-S
Processing
of
fusion
protein
SBT-M
11 1
0.80.8
fraction processed
fraction
fractionprocessed
processed
0.80.8
0.60.6
0.60.6
fraction
fraction
fraction
processed
processed
processed
S190
0.40.4
0.40.4
0.2
0.20.2
0.2
00 0
0 00 0
0
2
0.5
0.50.5
4
0.5
6
11 1
8
1
hours
hours
hours
hours
hours
10
1.5
1.51.5
12
1.5
14
22 16
2
2
S189 S190 vs pR8 FKAM
Processing
ofoffusion
protein
byby
SBT-S
Processing
fusion
protein
SBT-S
1
11
0.80.8
fraction processed
0.8
0.6
fraction
0.6 0.6
fraction
processed
processed
fraction
processed0.4
0.4 0.4
0.2
0.2
0.2 0
S189
-0.2 0
0 00
0
22
2
44
4
66
6
88
8
hours
hours
hours
1010
10
1212
12
1414
14
1616
16
Precise fusion of target protein onto pro-region
Target protein
ProR58
Nde I
Eco R1
Hind III
Sal I
Expression Vector pG58
Synthesis of fusion protein
Cell extract
Fusion protein
Binding of fusion protein to Sbt column
Loading on column
Flow-through
from column
Elution of processed target protein
Elution after overnight
incubation
Processed target protein
Purification of fusion protein
Acid elution No incubation
Fusion protein
Pro-domain proR58 stripped from column
with 0.1M H3PO4 pH 2.1
Purified proteins
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•
•
•
•
•
•
•
•
Streptococcal protein GB domain
Streptococcal protein Ga domain
Protein GB mutant G311
Staphylococcal Protein AB domain
Protein AB mutant A219
E. coli hypothetical Yab
Bovine a-subunit of transducin
M. thermautotrophicus CDC6
A. victoria Green Fluorescent Protein
56 aa
45 aa
56 aa
56 aa
56 aa
117 aa
350 aa
379 aa
238 aa
Conformational switching
A219
G311
25˚C
15N
HSQC spectra
G311
2˚C
a-subunit bovine transducin (350aa)
load
1
Fractions
2 3 4 5 6
pooled
a-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
• 0-1M Gu-HCl
(folding on the column)
Column is tolerant of:
•
•
•
•
EDTA
PMSF
Protease inhibitor cocktails
Reducing agents
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•
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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