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
Pichia
pre-pro-α-factor
signal seq.
Pichia
no added
signal seq.
E. coli
pelB signal
sequence
E. coli
no added
signal seq.

4. 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
LIC Site-ATG
STOP
LIC Site

5. Trials 1 Membrane ORF Target 2 12 24 96 (1) (2) (30) [1] [5] [7,680]
Full-length vs signal sequence truncation 2 12
LIC clone into 4 vectors- 2 clones each (E. coli storage strain)
Transform expression strain 24
E. coli (2 host strains)
Pichia (2 zeocin concentrations)
Small-scale expression tests in cell lysates
(vary temperature/induction time) 96
(1)
Intermediate-scale growth and membrane preparation
(2)
Cell fractionation (Membranes vs. Low speed pellet)
(30)
Solubility testing: ~15 detergents vs. low/high salt
[1]
Large-scale growth
[5]
Fractionation/Purification/Detergent exchange
[7,680]
Crystallization

6. Membrane Gel #21, Best Expressors; 10-3-03
Predicted Transmembrane ORFs Exhibiting High Expression in E. coli
183
114 81 64 50 37 26 21 15
8.4
kDa
Marker, 15 uL
Vector 21
(3) [10 kDa]
(4) [10 kDa]
(3) [12 kDa]
(4) [12 kDa]
(1) [20 kDa]
(2) [12 kDa]
(1) [27 kDa]
(2) [27 kDa]
(1) [35 kDa]
(2) [35 kDa]
(1) [38 kDa]
(1) [12 kDa]
(2) [12 kDa]
(2) [38 kDa]
(1) [21 kDa]
(2) [21 kDa]
Membrane Gel #21, Best Expressors;

7. Expression of L. major ORFs in SDS lysates
of E. coli BL21(DE3) Codon plus

8. Detergent Solubilization and IMAC Purification
Lmaj00191: Mitochondrial ADP/ATP Translocator
Total
Marker
Elution #4
Elution #1
Total
Elution #1
Talon Super
Wash #1
Elution #2
Elution #4
Elution #3
Elution #2
Elution #1
High Sp Pellet
Elution #3
High Sp Super
Equivalent of 0.3 ml culture
Equivalent of 1.5 ml culture
7.5 ml
Gel #60 [ (1)](Coomassie) –

9. (Acylphosphocholine) Detergents
Membrane protein purification from E. coli
Grow cells, induce, harvest, lyse (Avestin)
Low speed centrifugation
Pellet
Supernatant
Fos-choline
(Acylphosphocholine) Detergents
High speed centrifugation
Pellet
Supernatant OR
Treat pellet w/ 0.5% Fos-choline-16
Pellet
Supernatant
Concentration
Load on IMAC
Cleave on column
with His6-3C protease
exchange detergent
elute with imidazole
Rebind to IMAC
Ion Exchange
Dialysis

10. 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
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
(Equivalent of 0.4 ml culture)
(Equivalent
of 2.3 ml culture)

11. 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

12. 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 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 -
185 -
115 -
84 -
61 -
55 -
36 -
31 -
185 -
115 -
84 -
61 -
55 -
36 -
31 -
Immunoblot:
anti-RGSHis6
Immunoblot: anti-PGP
Immunoblot: anti-RGSHis6

13. 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

14. Vectors for Tetrahymena expression
“Soluble” 3C
Protease Site
Metallothionein promoter
RGS-6His
Calmodulin
Binding
Peptide
ATG-Cleavable signal
ORF
STOP
LIC Site
LIC Site
MADE
Membranes
“Soluble” 3C
Protease Site
Metallothionein promoter
RGS-6His
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
ATG
STOP

15. 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)

16. Rochester Membrane Protein Unit Not shown: Katrina Robinson
Back:
Kathy Clark, Earl Walker,
Mark Dumont
Front: Nadia Fedoriw
Not shown:
Katrina Robinson
Ina Urbatsch (Texas Tech)
Sara Connelly
Wim Hol