Expression and Purification of Membrane Proteins from Pathogenic Protozoa for Structural Genomics. Center for Human Genetics and Molecular Pediatric Disease.

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 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

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

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)

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

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

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

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

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

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

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

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

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

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)

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