Justification for producing proteins in a eukaryotic host - limitations of expression in E. coli

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

2. OBJECT: Develop tools to produce proteins for structural analysis in the yeast S. cerevisiae; emphasis on soluble protein complexes 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

3. MORF collection: A genomic array of ORF expression plasmids in yeast designed for protein purification PGAL1 new Tag: H6 HA 3c ZZ ORF attB attB’ Features: • Highly regulated control (PGAL) • Extensive sequence verifiication • Clonal: single plasmid (E. coli) and yeast • C terminal tag Control time of expression Analyze expression in yeast Functional membrane proteins

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 Martha Wilkinson Mike White Eric Phizicky, Mark Dumont, Mike Snyder, Dan Gelperin

5. H6-3C-10g H6-3C-2g MW -0.4g TKL1 HS ARO8 RAD6 TKL1 LYS1 TPD3 ALA1 MET22 HS SAM1 HS CKA1 ADE12 MET22 APN2 SAM1 URA7 ENO1 SOD1 LYS2 10 g 2 g IgG IgG Expression & Purification from yeast sufficient for X-ray crystallography attB PGAL attBORF HA ZZdomain His6 3C 2 URA3 MORF LEVLFQ/GPGP Yield: up to 0.5 mg/liter at OD = 1

6. Steps in development of yeast as an expression host 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. Vectors for High Level Expression of Affinity Tagged Proteins ORF1-3C-HA-H6-ZZ ORF2 (untagged) ORF1-3C-HA-H6-ZZ His6-ORF2 ORF1-3C-HA-H6-ZZ His10-ORF2 A Suite of LIC-LIC vectors to express up to 4 proteins per cell 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

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

9. Good yield and purity of yeast protein complexes. Trm112/Trm9 complex Purification: IgG-Talon-Sizing Trm9 Yield: 10.2 mg from 22 liters Trm112 MW, 0.4ug 5 ug 50 ug 15 ug 3CHis6, 5ug

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

12. 70 met peptide selmet peptide 50 Peptides relative abundance 20 0 0 0.1 0.2 0.5 0.375 [Selenomethionine] mM Selenomethionine substitution works in this strain Loss of met peptide with increasing selenomethionine concentration Appearance of corresponding selmet peptide Met Peptide:LNSANLMVVNHDAQFFPR Alan Friedman

13. MAD phasing works with proteins made in this strain: Structure of Wrs1p (tryptophan tRNA synthetase) solved with MAD. 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

14. Steps in development of yeast as an expression host 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. soluble insoluble Limitations of E. coli - solubility Expression & solubility of T Brucei ORFs expressed in E. coli expression (SDS lysates) solubility (crude extracts) MW markers

17. Test yeast as an expression host for heterologous genes Does expression in yeast correct solubility problem? Approach: Examine expression in yeast of L. major ORFs previously examined in E. Coli

18. Expression & solubility of L. major ORFs in yeast. Detection of L major ORFs in Crude Extract by Western kDa 93.8 61.5 60 54.8 50 40 30 20 QB519A QB518A 55 58 79 67 81 QB516A QB516A QB517A QB517A QB518A QB519A QB520A QB520A Magic Mark RCT MW mix GREEN: Total Protein-Hot SDS RED: SolubleProtein

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

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

21. Major ORFS bind IgG sepharose - folded 116kd 97.4kd 66.2kd IgG 45.0kd 31.0kd IgG 21.5kd 14.4kd 6.5kd 6864 6586 7489 2759 5499 4487 6168 6598 8264 - Lmaj ID#:

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

23. Yields of 15 proteins within range for structure

24. Steps in development of yeast as an expression host 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. THANKS to: Erin Quartley Yoshiko Kon Mike Malkowski Eric Phizicky Frederick Buckner and Wim Hol George deTitta Mark Dumont