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

2. 9th grade Biology text

3. 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)

4. 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

5. 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.

6. 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 signal sequence E. coli no added signal seq. Single PCR product

7. Expression of L. major ORFs in SDS lysates of E. coli BL21(DE3) Codon plus Target selection: 3 separate groups selected for: 1) known enzymes 2) small size 3) diversity (random selection)

8. Evolution of a Strategy for Membrane Protein Exprssion/Purification Current StrategySteps addedSteps eliminated Targeting of full-length and signal sequence-truncated ORFs Cloning into multiple vectorsCloning into vector w/ 3C cleavable signal Transforming into Pichia Use of multiple E coli strains Transforming into expression host Transforming into Tetrahymena Testing different temperaturesSmall scale expression at 25 o C Membrane fractionation Screening 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

9. Lmaj000817T 586 5_C0586 Potassium chloride KCl 0.1 M CAPS 0.1 M pH 10 PEG 4000 20% (w/v)

10. 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

11. (20% PEG 4000 0.1 M MgCl 2 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

13. 16405 -14213 2191 PelB signal sequence: 2210 Daltons 14213 16405 Partial removal of PelB signal sequence from Lmaj007473T MALDI-TOF

14. MALDI-TOF of Lmaj000817TAAA 13748 (13562 expected w/ signal) Expected 11352 for cleaved

15. PelB-containing LIC vectors (Insert Region) LIC Site 3C Protease Site ORF RGS-6His Calmodulin Binding Peptide STOP LIC Site ATG-Cleavable signal LIC Site 3C Protease Site ORF 6His STOP LIC Site ATG-pelB signal PSGP21: PelB + Cleavable C-terminal tags pSGP35: 3C cleavable PelB + N-terminal 6His

16. MALDI-TOF of purified ORFs expressed in pSGP35 (cleavable PelB) L5701 Lmaj004776T

17. 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.

18. 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 University Ted Clark, Cornell University Jacek Gaertig, University of Georgia Martin Gorovsky, University of Rochester

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

20. Targets for membrane and secreted protein expression in Tetrahymena: LocusFunction/Target rationaleAAs MAL7P1.27pfcrt (choroquine resistance) 424 PFE1150wmdr1 (drug resist)1419 PFE1265wGPCR? 467 Pf13_0248pf47 antigen 439 PFB0405s230 antigen3135 AAF63684.1pfs25 antigen 217 AAT00624.1pfs28 antigen 218 PF11_0486MAEBL eryth binding antigen2055 PFA0125cEbl1 erythrocyte binding antigen1567 PFL1315wpfkch1 K+channel1979 PFI0955wput. Hexose transporter 476 PFA0310cCa+ATPase (atemisinin target)1228

21. 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