Expression and Purification of Membrane Proteins from Pathogenic Protozoa for Structural Genomics.

1
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
2
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

3
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

Single PCR product
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E. coli and Pichia LIC vectors (Insert Region)
3C Protease Site
RGS-6His
Calmodulin Binding Peptide
ATG-Cleavable signal
ORF
STOP
LIC Site
LIC Site
3C Protease Site
RGS-6His
Calmodulin Binding Peptide
ORF
STOP
LIC Site-ATG
LIC Site
5
Trials
Membrane ORF Target Full-length vs signal
sequence truncation
1
2
12
LIC clone into 4 vectors- 2 clones each (E. coli
storage strain)
Transform expression strain
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E. coli (2 host strains)
Pichia (2 zeocin concentrations)
Small-scale expression tests in cell
lysates (vary temperature/induction time)
96
Intermediate-scale growth and membrane preparation
(1)
Cell fractionation (Membranes vs. Low speed
pellet)
(2)
Solubility testing 15 detergents vs. low/high
salt
(30)
1
Large-scale growth
5
Fractionation/Purification/Detergent exchange
7,680
Crystallization
6
Predicted Transmembrane ORFs Exhibiting High
Expression in E. coli
183
114
81
64
50
37
26
21
15
8.4
kDa
Vector 21
191-21-(2) 35 kDa
Marker, 15 uL
544-21-(3) 10 kDa
813-21-(1) 27 kDa
813-21-(2) 27 kDa
191-21-(1) 35 kDa
196-21-(1) 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
1197-21-(1) 12 kDa
196-21-(2) 38 kDa
1197-21-(2) 12 kDa
1235-21-(1) 21 kDa
1235-21-(2) 21 kDa
Membrane Gel 21, Best Expressors 10-3-03
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Expression of L. major ORFs in SDS lysates of E.
coli BL21(DE3) Codon plus
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Detergent Solubilization and IMAC Purification
Lmaj00191 Mitochondrial ADP/ATP Translocator

• Total

• Elution 4

• Marker

• Elution 1

• Elution 1

• Total

• Elution 3

• Wash 1

• Elution 2

• Elution 4

• Talon Super

• Elution 2

• Elution 1

• Elution 3

• High Sp Pellet

• 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
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Membrane protein purification from E. coli
Grow cells, induce, harvest, lyse (Avestin)
Low speed centrifugation
Supernatant
Pellet
Fos-choline (Acylphosphocholine) Detergents
High speed centrifugation
Pellet
Supernatant
OR
Treat pellet w/ 0.5 Fos-choline-16
Pellet
Supernatant
Load on IMAC
Concentration
exchange detergent elute with imidazole
Cleave on column with His6-3C protease
Dialysis
Rebind to IMAC
Ion Exchange
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Lmaj00191-21-1 Solubilization w/ Fos-Choline
16 Purification in dodecylmaltoside with
on-column 3C protease cleavage
Coomassie-stained gel
3C protease
Uncleaved 191
Cleaved 191
Marker Solubilization Super after Talon Wash
1 Wash 2 Wash 3 Fraction 1 Fraction
2 Fraction 3 Talon Resin Fraction 1 Fraction
2 Fraction 3 3C Protease
Sample solubilized in 0.5 FC-16 Protein cleaved
on Talon in 0.1 DDM and 0.05 PEG 3350 (Heimpel
et al. (2001) JBC 276, 11499)
KMC 3-11-04
(Equivalent of 0.4 ml culture)
(Equivalent of 2.3 ml culture)
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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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8/11 Human ABC genes express from SGPP Pichia
vectors at levels 1-10 times previous PGP level
(Ina Urbatsch)
P-Glycoprotein (PGP) in pHilD yields 10-20
mg/liter, purified from fermentor growth
pSGP17 -ABCF3
pSGP17
pSGP17
pSGP18
pSGP17 PGP(2)
pHilD PGP
pSGP18 ABCF2
pSGP17 ABCF2
pSGP18 ABCF3
pSGP17 PGP
185 -
115 -
84 -
61 -
55 -
36 -
31 -
Immunoblot anti-RGSHis6
Immunoblot anti-PGP
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 University Ted Clark,
Cornell University Jacek Gaertig, University of
Georgia Marty Gorovsky, University of
Rochester
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Vectors for Tetrahymena expression
Soluble 3C Protease Site
RGS-6His
Metallothionein promoter
Calmodulin Binding Peptide
ATG-Cleavable signal
ORF
STOP
LIC Site
LIC Site
MADE
Membranes
Soluble 3C Protease Site
RGS-6His
Metallothionein promoter
Calmodulin Binding Peptide
ORF
LIC Site-ATG
STOP
LIC Site
Under Construction
Metallothionein promoter
Soluble 3C Protease Site
ORF
6His
LIC Site
LIC Site
Soluble ORFs
MADE
STOP
ATG
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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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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