1. Engineering yeast to produce proteins for X-ray Crystallography: Heterologous Expression of L. MAJOR proteins in the yeast S. cerevisiae

2. Justification for producing proteins in a eukaryotic host - limitations of expression in E. coli - solubility - post-translational modifications of many eukaryotic proteins -advantage of S. cerevisiae for analysis of protein complexes - complexes best defined in yeast - homologous expression OBJECT: Develop tools to produce proteins for structural analysis in the yeast S. cerevisiae; emphasis on soluble protein complexes

3. Features: Highly regulated control (P GAL ) Extensive sequence verifiication Clonal: single plasmid (E. coli) and yeast C terminal tag ORF Tag: H6 HA 3c ZZ MORF collection: A genomic array of ORF expression plasmids in yeast designed for protein purification P GAL1 attBattB’ Analyze expression in yeast Control time of expression Functional membrane proteins new

4. Summary of MORF collection ORF targets:6,426 ORFs cloned and sent for sequencing 6,376 ORFs with correct sequence, two directions 5,854 (93.2%) fully sequenced ORFs 3,217 (55%) partially sequenced ORFs (~1100 bp ea) 2,637 (45%) Yoshiko Kon Mike White Martha Wilkinson Eric Phizicky, Mark Dumont, Mike Snyder, Dan Gelperin

5. Expression & Purification from yeast sufficient for X-ray crystallography LEVLFQ/GPGP 3C HAZZ domain 2  URA3 MORF P GAL attB ORF His6 attB IgG ARO8RAD6 TPD3 LYS1 TKL1 TKL1 HS H6-3C-10  g H6-3C-2  g MW -0.4  g CKA1 APN2 LYS2 ALA1 URA7 ENO1 MET22 HS SAM1 HS SOD1 MET22 ADE12 SAM1 2  g 10  g Yield: up to 0.5 mg/liter at OD = 1

6. Steps in development of yeast as an expression host 1.Developed vectors for high level expression, efficient purification and determination of protein interactions 2. Solved problem with selenomethionine incorporation in yeast to allow use of MAD phasing in yeast 3. Tested heterologous expression of L. major proteins in yeast - expression and solubility are good. (Direct evidence that expression in yeast resolves solubility issue for many proteins.)

7. Dual expression vectors feature Bi-directional GAL promoter: 2 ORFs expressed from each vector- different tags on each ORF Can express up to 4 ORFs per cell with 2 selectable markers A Suite of LIC-LIC vectors to express up to 4 proteins per cell Vectors for High Level Expression of Affinity Tagged Proteins ORF1-3C-HA-H6-ZZ ORF2 (untagged) His6-ORF2 His10-ORF2

8. ORF1-3C-HA-H6-ZZ His6-ORF2 His10-ORF2 Feature: co-purification indicates complex formation Different tags on ORF1 and ORF2 allow multistep affinity purification Step 1: IgG sepharose bind and elute with 3C protease Vectors with His6 (His 10) used to learn about co-purification Step 2: IMAC binding and elution with imidazole His 6 (10) available after IgG step

9. Trm112/Trm9 complex MW, 0.4ug 3CHis 6, 5ug 15 ug 50 ug 5 ug Trm112 Trm9 Purification: IgG-Talon-Sizing Yield: 10.2 mg from 22 liters Good yield and purity of yeast protein complexes.

10. Steps in development of yeast as an expression host 1.Developed vectors for high level expression, efficient purification and determination of protein interactions 2. Solved problem with selenomethionine incorporation in yeast to allow use of MAD phasing in yeast 3. Tested heterologous expression of L. major proteins in yeast - expression and solubility are good. (Direct evidence that expression in yeast resolves solubility issue for many proteins.) √

11. Methionine S-adenosylmethionine SAM1 SAM2 Delete SAM1 and SAM2 genes. X X - Mutants that do not convert methionine to S-adenosylmethionine grow on toxic levels of selenomethionine Blocking conversion of SeMet to S-adenosylSeMet solves the selenomethionine problem in yeast - Proteins are produced efficiently in sam1 - sam2 - mutants when grown in media with selenomethionine

12. Selenomethionine substitution works in this strain Met Peptide:LNSANL M VVNHDAQFFPR Loss of met peptide with increasing selenomethionine concentration [Selenomethionine] mM 00.2 0.375 0.5 Peptides relative abundance 0 20 50 70 met peptide 0.1 selmet peptide Appearance of corresponding selmet peptide Alan Friedman

13. Representative Electron Density for yeast WRS1. - MAD experimental electron density for Met-169, Met-174, & Met-360. -Three selenium atoms within Met side chains are clearly defined. Mike Malkowski MAD phasing works with proteins made in this strain: Structure of Wrs1p (tryptophan tRNA synthetase) solved with MAD.

14. Steps in development of yeast as an expression host 1.Developed vectors for high level expression, efficient purification and determination of protein interactions 2. Solved problem with selenomethionine incorporation in yeast to allow use of MAD phasing in yeast 3. Tested heterologous expression of L. major proteins in yeast - expression and solubility are good. (Direct evidence that expression in yeast resolves solubility issue for many proteins.) √ √

15. Rationale for producing proteins in a yeast is that many eukaryotic proteins are insoluble when expressed in E. coli

16. Limitations of E. coli - solubility Expression & solubility of T Brucei ORFs expressed in E. coli expression (SDS lysates) solubility (crude extracts) MW markers insoluble soluble

17. Does expression in yeast correct solubility problem? Test yeast as an expression host for heterologous genes Approach: Examine expression in yeast of L. major ORFs previously examined in E. Coli ClassNumber of ORFs Expression in E. coli Soluble protein in E. coli Test64GoodPoor Positive control 8Good Negative control11Poor

18. RCT MW mix Magic Mark QB517A QB518A QB519A QB520A 93.8 61.5 54.8 60 50 40 30 20 kDa 58796781 QB516A 55 Expression & solubility of L. major ORFs in yeast. GREEN: Total Protein-Hot SDS RED: Soluble Protein Detection of L major ORFs in Crude Extract by Western

19. TestPosNeg High Medium Low None Groups of L. major ORFs Number of ORFs 0 5 10 15 20 25 Most of the L major ORFs are expressed in yeast Pos = Positive control Neg = negative control

20. 0 % 20 % 60 % 100 % TestPosNeg Solubility of ORFs in Each Group Percent of ORFs Good solubility Insoluble Partial solubility Poor solubility Most test L major proteins are soluble in yeast Groups of L. major ORFs Pos = Positive control

21. - 6.5kd 14.4kd 21.5kd 31.0kd 45.0kd 66.2kd 97.4kd 116kd 448765988264 2759 6864 658674896168 5499 Lmaj ID#: L.Major ORFS bind IgG sepharose - folded IgG

22. 6976 Lmaj ID#: Purification of an L. Major ORF on IgG with 3C protease elution On IgG beads Post-1st IgG beads Post-2nd IgG beads 1st Elution 2nd Elution IgG Lyse cell Bind to IgG Cleavage with 3C Wash

23. Yields of 15 proteins within range for structure

24. Steps in development of yeast as an expression host 1.Developed vectors for high level expression, efficient purification and determination of protein interactions 2. Solved problem with selenomethionine incorporation in yeast to allow use of MAD phasing in yeast 3. Tested heterologous expression of L. major proteins in yeast - expression and solubility are good. (Direct evidence that expression in yeast resolves solubility issue for many proteins.) √ √ √

25. Erin QuartleyMike Malkowski Eric Phizicky George deTitta THANKS to: Yoshiko Kon Mark Dumont Frederick Buckner and Wim Hol