Expression and Purification of Membrane Proteins from Leishmania major for Structural Genomics.

Nadia Fedoriw, Kathy M. Clark, Sara M. Connelly, Katrina Robinson, Gayle Schneider, Wim G. Hol1, and Mark E. Dumont

Center for Human Genetics and Molecular Pediatric Disease and Department of Biochemistry and Biophysics. University of Rochester Medical Center. Rochester, NY 14642.

1Departments of Biochemistry and Biomolecular Structure, University of Washington, Seattle, Washington

9th grade Biology text

High resolution structures of transmembrane proteins (as of 1/05)

http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html

(Includes multi-species)

Bacterial helical membrane proteins: 26

Bacterial porin/β-barrel outer membrane proteins: 25

“Eukaryotic” helical membrane proteins: 3

Soluble structures in database (3/05) 29,956 (Source:http://www.rcsb.org/pdb/holdings_table.html)

Membrane Proteins: Initial 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

SGPP Membrane protein highlights: 3/2004-3/2005

1. Expression at 1 mg /liter of >40% of selected predicted transmembrane ORFs from L. major in an E. coli expression system.

2. Purification of 1-4 batches of 10 different L. Major predicted transmembrane proteins for crystallization

3. Detection of possible crystal hits for two ORFs at HWI

4. Confirmation of crystal hits for one ORF in optimizations set up in Rochester:

5. Partial or complete retention of PelB signal sequence in current vectors.

6. Construction and testing of a new vector allowing quantitative pelB cleavage.

7. Construction of vectors and cloning of P. falciparum ORFs for expression in Tetrahymena.

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

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

PelB signal(Total Set)(pSGP21)

PelB signalSmall ORFS(<300 AAs)(pSGP21)

No signal(Total Set) (pSGP22)

Number of tested targets 126 42 54

Clones with detectable expression(Immunoblotting) 106 (84%) 39 (93%) 28 (52%)

Clones with detectable expression(Coomassie staining) 51 (40%) 32 (76%) 4 (7%)

Target selection: 3 separate groups selected for:1) known enzymes2) small size 3) diversity (random selection)

Evolution of a Strategy for Membrane Protein Exprssion/Purification

Current Strategy Steps addedSteps eliminatedTargeting of full-length and

signal sequence-truncated ORFs

Cloning into multiple vectors Cloning into vector w/ 3C cleavable signal

Transforming into PichiaUse of multiple E coli strains

Transforming into expression host

Transforming into Tetrahymena

Testing different temperatures Small scale expression at 25oC

Membrane fractionationScreening of initial detergent

Fos choline-16 extraction of whole-cell extract

IMAC affinity - detergent exchange - 3C protease elution

6-18 liter cultures

Gel Filtration - Centricon concentration

~1 liter optimization

Lmaj000817T

5865_C0586Potassium chloride KCl 0.1 MCAPS 0.1 M pH 10PEG 4000 20% (w/v)

E4 0.1M Tris, pH 8.5 0.1M MgSO4 PEG 4000, 15% E4 0.1M Tris, pH 8.5 0.1M MgSO4 PEG 4000, 15%

E7 0.1M Tris, pH 8.5 0.1M KCl PEG 4000, 15% D4 0.1M Tris, pH 8.0 0.1M MgSO4 PEG 4000, 15%

Crystallizations from 1% dodecylmaltoside (4 weeks) Lmaj000817T

(20% PEG 4000 0.1 M MgCl2 0.1M Tris pH 8.5)

Crystallization from 1% dodecylmaltoside (4 weeks)

Crystallization from 0.8% dodecylmaltoside (12 days)

0.1 M Hepes pH 8.3, 0.1 M MgCl2, 21% PEG 4000

Lmaj000817T

16405-14213 2191

PelB signal sequence:2210 Daltons14213

16405

Partial removal of PelB signal sequence from Lmaj007473T

MALDI-TOF

MALDI-TOF of Lmaj000817TAAA13748

(13562 expected w/ signal)

Expected 11352 for cleaved

PelB-containing LIC vectors(Insert Region)

LIC Site

3C ProteaseSite

ORF

RGS-6HisCalmodulin

Binding Peptide STOP

LIC Site

ATG-Cleavable signal

LIC Site

3C ProteaseSite

ORF6His

STOPLIC Site

ATG-pelB signal

PSGP21: PelB + Cleavable C-terminal tags

pSGP35: 3C cleavable PelB + N-terminal 6His

MALDI-TOF of purified ORFs expressed in pSGP35 (cleavable PelB)

L5701 Lmaj004776T

Crystallization strategies

1. Optimization for PelB-containing Lmaj000817T- frozen crystals to be shipped or carried (Katrina Robinson) to Seattle Decreasing detergent concentration Cryopreservation Detergent mixes Additives Temperature

2. Production of antibodies to Lmaj000817T (ongoing, w/ Mark Sullivan)

3. Purification of Lmaj000817T lacking PelB from pSGP35.

4. Revisiting purification of other good expressors that purify as homogeneous protein containing PelB.

5. Targeting ORFs that express well in pSGP35 lacking PelB

6. Construction of a new vector: PelB-High expressing ORF-His 6-3C site.

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

3. Evolutionary relatedness of Tetrahymena to 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 Martin 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

“Soluble” 3CProtease SiteMembranes

LIC Site

“Soluble” 3C Protease SiteORF6His

STOP

LIC Site

ATG

Metallothionein promoter

Soluble ORFs

Targets for membrane and secreted protein expression in Tetrahymena:

Locus Function/Target rationale AAs

MAL7P1.27 pfcrt (choroquine resistance) 424PFE1150w mdr1 (drug resist) 1419PFE1265w GPCR? 467Pf13_0248 pf47 antigen 439PFB0405 s230 antigen 3135AAF63684.1 pfs25 antigen 217AAT00624.1 pfs28 antigen 218PF11_0486 MAEBL eryth binding antigen 2055PFA0125c Ebl1 erythrocyte binding antigen 1567PFL1315w pfkch1 K+channel 1979PFI0955w put. Hexose transporter 476PFA0310c Ca+ATPase (atemisinin target) 1228

Kathy Clark, Nadia Fedoriw, Katrina Robinson, Gayle Schneider, Sara Connelly

Thanks to:

Eric Phizicky, Elizabeth Grayhack, Mark Sullivan

Ina Urbatsch (Texas Tech Medical Center)

Michael Malkowski (HWI)

Jolanta Kruczinska, Joseph Wedekind, (Crystallography- Rochester)

Edward Petri (laboratory of Ravi Basavappa) (Crystallography- Rochester)

Tetragenetics

Rochester Membrane Protein Unit