expression and purification of membrane proteins from pathogenic protozoa for structural genomics....
TRANSCRIPT
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Expression and Purification of Membrane Proteins from Pathogenic Protozoa for Structural Genomics.
Center for Human Genetics and Molecular Pediatric Disease and Department of Biochemistry and Biophysics.
University of Rochester Medical Center, Rochester, NY
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Membrane Proteins: Strategies
1. Trypanosomatids only (initial)
2. 2 predicted transmembrane segments
3. Expression in Pichia Pastoris and E. coli
4. Ligation-Independent cloning into C-terminal cleavable double-tagged vector
5. Purified protein to be sent for crystallization in a small number of crystallography-proven detergents (~5)
6. Co-crystallization with single chain antibodies and two-hybrid binding partners
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Cloning Strategy for Membrane Protein Expression
Use ligation independent cloning to insert a single PCR-product into two E. coli vectors and two Pichia vectors
Pichia pre-pro-α-factor
signal seq.
Pichia no added signal seq.
E. coli pelB signalsequence
E. coli no addedsignal seq.
Single PCR product
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LIC Site-ATG ORF LIC Site
E. coli and Pichia LIC vectors(Insert Region)
LIC Site
3C ProteaseSite
ORF
RGS-6HisCalmodulin
Binding Peptide STOP
LIC Site
ATG-Cleavable signal
3C ProteaseSite RGS-6His
Calmodulin Binding Peptide STOP
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Membrane ORF TargetFull-length vs signal sequence truncation
LIC clone into 4 vectors- 2 clones each (E. coli storage strain)
Transform expression strainE. coli (2 host strains) Pichia (2 zeocin concentrations)
Small-scale expression tests in cell lysates(vary temperature/induction time)
Intermediate-scale growth and membrane preparation
Solubility testing: ~15 detergents vs. low/high salt
Large-scale growth
Crystallization
Fractionation/Purification/Detergent exchange
Cell fractionation (Membranes vs. Low speed pellet)
1
12
24
96
(1)
(30)
[1]
[5]
Trials
[7,680]
2
(2)
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Membrane Gel #21, Best Expressors; 10-3-03
Marker, 15 uL
Vector 21
544-21-(3) [ 10 kDa]
1197-21-(1) [12 kDa]
1197-21-(2) [12 kDa]
1235-21-(1) [21 kDa]
1235-21-(2) [21 kDa]
813-21-(1) [ 27 kDa]
813-21-(2) [ 27 kDa]
191-21-(1) [35 kDa]
191-21-(2) [35 kDa]
196-21-(1) [38 kDa]
196-21-(2) [ 38 kDa]
495-21-(3) [ 12 kDa]
495-21-(4) [ 12 kDa]
751-21-(1) [ 20 kDa]
817-21-(2) [12 kDa]
544-21-(4) [ 10 kDa]
183114
816450
37
26
21
15
8.4
kDa
Predicted Transmembrane ORFs Exhibiting High Expression in E. coli
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PelB signal(pSGP21)
No signal sequence(pSGP22)
Number of tested targets 84 54
Clones with detectable expression(Immunobloting)
67 28
Clones with detectable expression(Coomassie staining)
19 4
Expression of L. major ORFs in SDS lysates of E. coli BL21(DE3) Codon plus
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Gel #60 [191-21-(1)](Coomassie) –11-25-03
Marker
Total
High S
p Super
High S
p Pellet
Talon S
uper
Wash #1
Elution #1
Elution #2
Elution #3
Elution #4
Total
Elution #1
Elution #2
Elution #3
Elution #4
Elution #1
Detergent Solubilization and IMAC Purification Lmaj00191: Mitochondrial ADP/ATP Translocator
Equivalent of 0.3 ml culture Equivalent of 1.5 ml culture 7.5 ml
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Grow cells, induce, harvest, lyse (Avestin)
Low speed centrifugation
Treat pellet w/ 0.5% Fos-choline-16
High speed centrifugation
Load on IMAC
Cleave on column with His6-3C protease
Supernatant
Pellet
exchange detergentelute with imidazole
Membrane protein purification from E. coli
Pellet
Pellet
Supernatant
Supernatant
OR
DialysisIon Exchange
Concentration
Fos-choline (Acylphosphocholine) Detergents
Rebind to IMAC
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Lmaj00191-21-1: Solubilization w/ Fos-Choline 16Purification in dodecylmaltoside with on-column 3C protease cleavage
Mar
ker
Sol
ubi
lizat
ion
Sup
er a
fter
Tal
on
Was
h 1
Was
h 2
Was
h 3
Fra
ctio
n 1
Fra
ctio
n 2
Fra
ctio
n 3
Tal
on R
esi
n
Fra
ctio
n 1
Fra
ctio
n 2
Fra
ctio
n 3
3C P
rote
ase
Sample solubilized in0.5% FC-16
Protein cleaved on Talonin 0.1% DDM and 0.05% PEG 3350(Heimpel et al. (2001) JBC 276, 11499)
KMC 3-11-04
3C protease
Uncleaved 191Cleaved 191
(Equivalent of 0.4 ml culture) (Equivalent of 2.3 ml culture)
Coomassie-stained gel
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Expression Summary: Pichia
(6 ORFs expressed in both vectors)
Typical expression level (from calibrated Westerns):
0.5 mg per 8 g wet cells grown in 1 liter shaking culture
Fermentor growth of Pichia 400 g cells/liter 25mg protein/liter
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185 -115 - 84 -
61 - 55 -
36 -31 -
pSGP17PGP(2)
pS
GP
17
pHilDPGP
(Ina Urbatsch)
Immunoblot: anti-PGP
P-Glycoprotein (PGP) in pHilD yields 10-20 mg/liter, purified from fermentor growth
8/11 Human ABC genes express from SGPP Pichia vectors at levels 1-10 times previous PGP level
185 -115 - 84 -
61 - 55 -
36 -31 -
pSGP18ABCF2
pSGP17ABCF2
pS
GP
17
pS
GP
18
pSGP18ABCF3
pS
GP
17
-AB
CF
3
185 -115 - 84 - 61 - 55 -
36 -31 -
pSGP17PGP
Immunoblot: anti-RGSHis6 Immunoblot: anti-RGSHis6
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Tetrahymena as a host for expression of membrane proteins from Plasmodium falciparum
Advantages:
1. High membrane content coating abundant cilia.
2. High genomic AT content, may be beneficial for expressing P. falciparum genes
3. Tetrahymena is a protozoan, like P. falciparum
4. Recently developed as a genetic system (Gaertig, Gorovsky et al.)
Collaborators: Tetragenetics Inc:Donna Cassidy-Hanley, Cornell UniversityTed Clark, Cornell UniversityJacek Gaertig, University of Georgia Marty Gorovsky, University of Rochester
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Vectors for Tetrahymena expression
LIC Site-ATG ORFLIC Site
LIC Site
“Soluble” 3CProtease Site
ORF
RGS-6His
Calmodulin Binding Peptide STOP
LIC Site
ATG-Cleavable signal
RGS-6HisCalmodulin
Binding Peptide STOP
Metallothionein promoter
Metallothionein promoter
MADE
Under Construction
“Soluble” 3CProtease SiteMembranes
LIC Site
“Soluble” 3C Protease SiteORF6His
STOP
LIC Site
ATG
Metallothionein promoter
MADESoluble ORFs
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Conclusions
1. A surprising number of Leishmania membrane proteins express to high levels in E. coli and Pichia
2. Heterologously expressed Leishmania membrane proteins are resistant to solubilization with most common detergents.
3. Leishmania membrane proteins can be purified in a small number of steps from E. coli and exchanged into suitable detergents.
4. It will be important to use an initial set of targets that can be assayed for function.
5. Every protein is different. (Expression, Solubility, Susceptibility to cleavage, Prokaryotes vs. Eukaryotes)
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Back: Kathy Clark, Earl Walker,Mark Dumont
Front: Nadia Fedoriw
Not shown:
Katrina Robinson Ina Urbatsch (Texas Tech)
Sara Connelly
Rochester Membrane Protein Unit
Wim Hol