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Engineering yeast to produce proteins for X-ray Crystallography: Heterologous Expression of L. MAJOR proteins in the yeast S. cerevisiae. OBJECT: Develop tools to produce proteins for structural analysis in the yeast S. cerevisiae ; emphasis on soluble protein complexes.

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slide1

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

slide2

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

slide3

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

slide4

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

slide5

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

slide6

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.)

slide7

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

slide8

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

slide9

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

slide10

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.)

slide11

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

slide12

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

slide13

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

slide14

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.)

slide15

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

slide16

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

slide17

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

slide18

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

slide19

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

slide20

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

slide21

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#:

slide22

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

slide24

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.)

slide25

THANKS to:

Erin Quartley

Yoshiko Kon

Mike Malkowski

Eric Phizicky

Frederick Buckner and Wim Hol

George deTitta

Mark Dumont