Transcript PPT - CABM Protein NMR Lab

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
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
Cloning Strategy for Membrane Protein Expression
Use ligation independent cloning to insert a single PCRproduct into two E. coli vectors and two Pichia vectors
Single PCR product
Pichia
Pichia
E. coli
E. coli
pre-pro-α-factor
signal seq.
no added
signal seq.
pelB signal
sequence
no added
signal seq.
E. coli and Pichia LIC vectors
(Insert Region)
3C Protease
Site
ATG-Cleavable
signal
LIC Site
LIC Site-ATG
ORF
ORF
LIC Site
RGS-6His
Calmodulin
Binding
STOP
Peptide
3C Protease
Site
RGS-6His
Calmodulin
Binding
STOP
Peptide
LIC Site
Trials
1
2
12
24
96
(1)
Membrane ORF Target
Full-length vs signal sequence truncation
LIC clone into 4 vectors- 2 clones each (E. coli storage strain)
E. coli (2 host strains)
Transform expression strain
Pichia (2 zeocin concentrations)
Small-scale expression tests in cell lysates
(vary temperature/induction time)
Intermediate-scale growth and membrane preparation
(2)
Cell fractionation (Membranes vs. Low speed pellet)
(30)
Solubility testing: ~15 detergents vs. low/high salt
[1]
[5]
[7,680]
Large-scale growth
Fractionation/Purification/Detergent exchange
Crystallization
Predicted Transmembrane ORFs Exhibiting High Expression in E. coli
183
114
81
64
50
37
26
21
15
8.4
kDa
Membrane Gel #21, Best Expressors; 10-3-03
Expression of L. major ORFs in SDS lysates
of E. coli BL21(DE3) Codon plus
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
Detergent Solubilization and IMAC Purification
Lmaj00191: Mitochondrial ADP/ATP Translocator
 Elution #1
 Elution #4
 Elution #3
 Elution #2
 Elution #1
 Total
 Elution #4
 Elution #3
 Elution #2
 Elution #1
 Wash #1
 Talon Super
 High Sp Pellet
Total
 High Sp Super

Equivalent of 0.3 ml culture
Equivalent of 1.5 ml culture
7.5 ml
Gel #60 [191-21-(1)](Coomassie) –11-25-03
Membrane protein
purification from E. coli
Grow cells, induce, harvest, lyse (Avestin)
Low speed centrifugation
Fos-choline
(Acylphosphocholine) Detergents
Pellet
Supernatant
High speed centrifugation
OR
Pellet
Supernatant
Treat pellet w/ 0.5% Fos-choline-16
Pellet
Load on IMAC
Concentration
Cleave on column
with His6-3C protease
Rebind to IMAC
Supernatant
Ion Exchange
exchange detergent
elute with imidazole
Dialysis
Lmaj00191-21-1: Solubilization w/ Fos-Choline 16
Purification in dodecylmaltoside with on-column 3C protease cleavage
Coomassie-stained gel
3C protease
Uncleaved 191
Protein cleaved on Talon
in 0.1% DDM and
(Equivalent of 0.4 ml culture)
0.05% PEG 3350
(Heimpel et al. (2001) JBC 276, 11499)
(Equivalent
of 2.3 ml culture)
3C Protease
Fraction 3
Fraction 2
Fraction 1
Talon Resin
Fraction 3
Fraction 2
Fraction 1
Wash 3
Wash 2
Wash 1
Super after Talon
Sample solubilized in
0.5% FC-16
Solubilization
Marker
Cleaved 191
KMC
3-11-04
Expression Summary: Pichia
Vector 17
(pp- signal)
Vector 18
(no signal)
10/20
10/15
Truncated
3/3
3/3
Full length
7/17
7/12
Extra bands
7/20
4/15
>2
-
Total
Incomplete signal cleavage
(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
8/11 Human ABC genes express from SGPP Pichia vectors
at levels 1-10 times previous PGP level
(Ina Urbatsch)
pSGP17
PGP(2)
pHilD
PGP
pSGP18
ABCF2
185 115 84 61 55 -
185 115 84 61 55 -
36 31 -
36 31 -
Immunoblot: anti-PGP
pSGP17
ABCF2
pSGP17
-ABCF3
pSGP17
pSGP18
pSGP17
P-Glycoprotein (PGP) in pHilD yields 10-20 mg/liter, purified from fermentor growth
pSGP18
ABCF3
pSGP17
PGP
185 115 84 61 55 36 31 -
Immunoblot: anti-RGSHis6
Immunoblot:
anti-RGSHis6
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 University
Ted Clark, Cornell University
Jacek Gaertig, University of Georgia
Marty Gorovsky, University of Rochester
Vectors for Tetrahymena expression
“Soluble” 3C
Protease Site
Metallothionein promoter
ATG-Cleavable signal
LIC Site
Membranes
ORF
Metallothionein promoter
LIC Site-ATG
ORF
LIC Site
RGS-6His
Calmodulin
Binding
Peptide
STOP
“Soluble” 3C
RGS-6His
Protease Site
Calmodulin
Binding
Peptide
STOP
LIC Site
Metallothionein promoter
“Soluble” 3C Protease Site
ORF
6His
LIC Site
LIC Site
Soluble ORFs
ATG
STOP
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)
Rochester Membrane
Protein Unit
Back:
Kathy Clark, Earl Walker,
Mark Dumont
Front: Nadia Fedoriw
Not shown:
Katrina Robinson
Ina Urbatsch (Texas Tech)
Sara Connelly
Wim Hol