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
ORF
Tag: H6 HA 3c ZZ
new
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: ,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. Yield: up to 0.5 mg/liter at OD = 1
Expression & Purification from yeast sufficient for
X-ray crystallography
PGAL attB ORF
attB
His6 HA 3C
ZZdomain
MORF
2 URA3
LEVLFQ/GPGP
Yield: up to 0.5 mg/liter at OD = 1
IgG
ARO8
RAD6
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

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
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
ORF1-3C-HA-H6-ZZ
ORF2 (untagged)
His6-ORF2
His10-ORF2

8. Vectors with His6 (His 10) used to learn about co-purification
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
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
15 ug
50 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. X X 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. Selenomethionine substitution works in this strain
Loss of met peptide with increasing selenomethionine concentration
Appearance of corresponding selmet peptide
[Selenomethionine] mM
0.2
0.375
0.5
Peptides relative abundance 20 50 70
met peptide
0.1
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. Limitations of E. coli - solubility
Expression & solubility of T Brucei ORFs expressed in E. coli
expression
(SDS lysates)
insoluble
soluble
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
Class
Number of ORFs
Expression in E. coli
Soluble protein in E. coli
Test 64
Good
Poor
Positive control 8
Negative control 11

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
QB516A
QB516A 55
QB517A
QB517A 58
QB518A
QB518A 79
QB519A
QB519A 67
QB520A
QB520A 81
Magic Mark
RCT MW mix
GREEN: Total Protein-Hot SDS
RED: Soluble Protein

19. Most of the L major ORFs are expressed in yeast5 10 15 20 25
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
45.0kd
IgG
31.0kd
IgG
21.5kd
14.4kd
6.5kd
6864
2759
8264
4487
7489
6586
6598
6168
5499 -
Lmaj ID#:

22. Purification of an L. Major ORF on IgG with 3C protease elution
Lmaj ID#:
6976
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: Frederick Buckner and Wim Hol Eric Phizicky Erin Quartley
Yoshiko Kon
Mike Malkowski
Frederick Buckner and Wim Hol
George deTitta
Mark Dumont