Engineering yeast to produce proteins for X-ray

Crystallography: Heterologous Expression of L.

MAJOR proteins in the yeast S. cerevisiae

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

Features:

• Highly regulated control (PGAL)

• 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

PGAL1

attB attB’

Analyze expression in yeast

Control time of expression

Functional membrane proteins

new

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

Expression & Purification from yeast sufficient forX-ray crystallography

LEVLFQ/GPGP

3C HA ZZdomain 2 URA3MORF PGAL attB ORF His6 attB

IgG

IgG

AR

O8

RA

D6

TP

D3

LY

S1

TK

L1

TK

L1

HS

H6-

3C-1

0g

H6-

3C-2

g

MW

-0.

4g

CK

A1

AP

N2

LY

S2

AL

A1

UR

A7

EN

O1

ME

T22

HS

SA

M1

HS

SO

D1

ME

T22

AD

E12

SA

M1

2 g

10

g

Yield: up to 0.5 mg/liter at OD = 1

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.)

Dual expression vectors feature Bi-directional GAL promoter:2 ORFs expressed from each vector- different tags on each ORFCan 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

2 URA3

ORF2 ORF1-tag

Vectors for High Level Expression of Affinity Tagged Proteins

ORF1-3C-HA-H6-ZZ

ORF1-3C-HA-H6-ZZ

ORF1-3C-HA-H6-ZZ

ORF2 (untagged)

His6-ORF2

His10-ORF2

2 URA3CYC terminator ORF2 PGAL1,10 ORF1 3C site HA H6 ZZ2 URA3CYC terminator ORF2 PGAL1,10 ORF1 3C site HA H6 ZZ

2 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZ2 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZ

2 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis62 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis6

2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis6CYC terminator2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis6CYC terminator

2 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis102 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis10

2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis10CYC terminator2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis10CYC terminator

ORF1-3C-HA-H6-ZZ

ORF1-3C-HA-H6-ZZ

His6-ORF2

His10-ORF2

2 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis62 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis6

2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis6CYC terminator2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis6CYC terminator

2 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis102 LEU2CYC terminator ORF4 PGAL1,10 ORF3 3C site HA H6 ZZHis10

2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis10CYC terminator2 URA3 ORF2 PGAL1,10 ORF1 3C site HA H6 ZZHis10CYC terminator

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

Trm112/Trm9 complexM

W,

0.4u

g

3CH

is6,

5ug

15 u

g

50 u

g

5 ug

Trm112

Trm9Purification: IgG-Talon-Sizing

Yield: 10.2 mg from 22 liters

Good yield and purity of yeast protein complexes.

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.)

√

Methionine S-adenosylmethionineSAM1

SAM2

Delete SAM1 and SAM2 genes.

XX

XX- Mutants that do not convert methionine to S-adenosylmethionine grow on toxic levels of selenomethionine

Blocking conversion of SeMet to S-adenosylSeMetsolves the selenomethionine problem in yeast

- Proteins are produced efficiently in sam1-sam2- mutants when grown in media with selenomethionine

Selenomethionine substitution works in this strain

Met Peptide:LNSANLMVVNHDAQFFPR

Loss of met peptide with increasing selenomethionine concentration

[Selenomethionine] mM

0 0.2 0.375 0.5

Pep

tid

es r

elat

ive

abu

nd

ance

0

20

50

70

met peptide

0.1

selmet peptide

Appearance of corresponding selmet peptide

Alan Friedman

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.

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.)

√

√

Rationale for producing proteins in a yeast is that many

eukaryotic proteins are insoluble when expressed in E. coli

Limitations of E. coli - solubilityExpression & solubility of T Brucei ORFs expressed in E. coli

expression (SDS lysates)

solubility (crude extracts)

MW markers

insoluble

soluble

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

Class Number of ORFs

Expression in E. coli

Soluble protein in E. coli

Test 64 Good Poor

Positive control 8 Good Good

Negative control 11 Poor

RC

T M

W m

ix

Mag

ic M

ark

QB

517A

QB

517A

QB

518A

QB

518A

QB

519A

QB

519A

QB

520A

QB

520A

93.8

61.554.860

5040

30

20

kDa

58 79 67 81

QB

516A

QB

516A

55

Expression & solubility of L. major ORFs in yeast.

GREEN: Total Protein-Hot SDSRED: Soluble Protein

Detection of L major ORFs in Crude Extract by Western

Test Pos Neg

High

Medium

Low

None

Groups of L. major ORFs

Nu

mb

er

of

OR

Fs

0

5

10

15

20

25

Most of the L major ORFs are expressed in yeast

Pos = Positive control

Neg = negative control

0. 0%

20. 0%

40. 0%

60. 0%

80. 0%

100. 0%

120. 0%

A B C0 %

20 %

60 %

100 %

Test Pos Neg

Solubility of ORFs in Each Group

Pe

rce

nt

of

OR

Fs Good solubility

Insoluble

Partial solubilityPoor solubility

Most test L major proteins are soluble in yeast

Groups of L. major ORFs

Pos = Positive control

-

6.5kd

14.4kd

21.5kd

31.0kd

45.0kd

66.2kd97.4kd

116kd

4487

6598

8264

2759

6864

6586

7489

6168

5499

Lmaj ID#:

L. Major ORFS bind IgG sepharose - folded

IgG

IgG

6976Lmaj ID#:

Purification of an L. Major ORF on IgG with 3C protease elution

On

IgG

bea

dsP

ost-

1st I

gG b

eads

Pos

t-2n

d Ig

G b

eads

1st E

lutio

n

2nd

Elu

tion

IgG

IgG

Lyse cell

Bind to IgG

Cleavage with 3CWash

Wash

108100.0%High2-hydroxyacid dehydrogenase5499

84100.0%Highsmall G-protein, putative6168

140100.0%Highanion-transporting ATPase7489

96100.0%Highn-acyl-l-amino acid amidohydrolase4487

9683.3%Highbeta-fructosidase-like protein ,8264

160100.0%Highflagellar protofilament ribbon6864

12066.7%Highmethyltransferase, putative6679

120100.0%Highcaltractin, putative6598

100100.0%Highphenylalanyl-tRNA synthetase2759

16083.3%HighGMP synthase2393

1683.3%Highserine peptidase 4763

140100.0%Highglucokinase 1-like protein6586

24083.3%HighSAM decarboxylase proenzyme4089

120100.0%High'monoglyceride lipase, 4486

88100.0%Mediumsterol 24-c-methyltransferase, 6593

200100.0%Mediumcyclin 1,serine peptidase6976

Yield: g per OD liter

Percent soluble

Express-ion in yeastPutative function of protein

L majorORF

108100.0%High2-hydroxyacid dehydrogenase5499

84100.0%Highsmall G-protein, putative6168

140100.0%Highanion-transporting ATPase7489

96100.0%Highn-acyl-l-amino acid amidohydrolase4487

9683.3%Highbeta-fructosidase-like protein ,8264

160100.0%Highflagellar protofilament ribbon6864

12066.7%Highmethyltransferase, putative6679

120100.0%Highcaltractin, putative6598

100100.0%Highphenylalanyl-tRNA synthetase2759

16083.3%HighGMP synthase2393

1683.3%Highserine peptidase 4763

140100.0%Highglucokinase 1-like protein6586

24083.3%HighSAM decarboxylase proenzyme4089

120100.0%High'monoglyceride lipase, 4486

88100.0%Mediumsterol 24-c-methyltransferase, 6593

200100.0%Mediumcyclin 1,serine peptidase6976

Yield: g per OD liter

Percent soluble

Express-ion in yeastPutative function of protein

L majorORF

Yields of 15 proteins within range for structure

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.)

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√

√

Erin Quartley Mike MalkowskiEric Phizicky

George deTitta

THANKS to:

Yoshiko Kon

Mark Dumont

Frederick Buckner and Wim Hol