A facile method that allows high-throughput co-expression from plasmids with identical replication

origins and antibiotic resistance markers (University of Rochester)

SGPP

A convenient method to co-express protein pairs

(University of Rochester)

SGPP

Set of expression plasmids:

ORF B ORF E

Goal: To co-express two proteins using a set of existing plasmids:

+

1. Two plasmids, two compatible origins, two selection markers (Amp, Kan)

2. One plasmid: one promoter, polycistronic mRNA

3. One plasmid: two promoters, two ORFs

A new plasmid has to be made

?

ORF A

ORF B

ORF C

ORF D

ORF E

ORF A ORF B

ORF B

ORF A

+

ORF A ORF B

ORF B

ORF A

+

• Can it be made?

• Will it work?

ORF A ORF B

ORF B

ORF A

+

Ligation:

ORF A ORF B

ORF B

ORF A

+

Ligation:

12

34

1 2

34

1 anneals to 4 only

2 anneals to 3 only

ORF A ORF B

ORF B

ORF A

12

34

1 234

1 anneals to 4 only

2 anneals to 3 only

ORF A ORF B

ORF B

ORF A

ORFPromoter

Expression vector

FLIP(61bp)

(FLIP version)

1 anneals to 4 only

2 anneals to 3 only

ORF B ORF E

+

ORF B ORF E

+

ORF B

ORF E

12

34

1 2

4 3

ORF BORF E1 24 3

Co-expression of the desired protein pair from the set:

Set of expression plasmids:

ORF A

ORF B

ORF C

ORF D

ORF E

emptyvector

insert 4

insert 3

insert 2insert 1

2

4

6810

1

0.2

prot.4

12A

Ladd

er

1 1 +

2

1 +

3

1 +

4

2 +

3

2 +

4

3 +

4

2 3 4

Ladd

er

1 1 +

2

1 +

3

1 +

4

2 +

3

2 +

4

3 +

4

2 3 4

D

prot.2

prot.1

prot.3

21

14

6

31

45

66

97116

pLysS

C

Ladd

er

Ladd

er

1 1 +

2

1 +

3

1 +

4

2 +

3

2 +

4

3 +

4

2 3 4B

4

6

8

1012

4

6

8

1012

E3 +

E2

a b c d e f g h i j k

a b c d e f g h i j k a b c d e f g h i j k

200

Co-expression of all pair combinations for 4 proteins (L. major)(equivalent to 400 Liters of growth):

A. Tandem plasmid contains two inserts.

B. Tandem plasmid has double size.

C. Tandem plasmid propagates in expression cells.

D. Protein pairs are expressed: all combinations.

• It uses an existing set of ORFs in identical plasmids.

• No primers needed.

• No PCR used ---> No sequencing needed.

• No PCR-purification, no gel-purification.

• No ligation.

• Octamer restriction enzymes allow to “flip” > 99.1% of possible random protein pairs.

• No selectable markers or compatible origins to worry about.

• “FLIP” sequence (61 bp) does not harm, if not used.

Advantages of the FLIP method:

LIC-cloning

The Protocol:1) Digest with a restriction enzyme.

2) Heat inactivate the restriction enzyme (60o, 20min)

3) T4-treat

4) Anneal two plasmids (1 min) and transform into E. Coli.

P. falciparum pairs

Target pairs: obtained in S. Fields lab using yeast two-hybrid screen.

MW

, 0.4

ug

3C, 2

0ug

EQ

2995

B

EQ

2997

B

EQ

3005

B

EQ

3009

B

E

Q30

41B

EQ

3027

B

EQ

3023

B

EQ

3043

B

EQ

3047

B E

Q30

61B

EQ

3063

B

EQ

3071

B

EQ

3075

B

EQ

3093

B

EQ

3089

B

EQ

3107

B

C1a C2a C6a C8a D3a D5a D12a E1a E3a C1b C2b C6a C8b D3b D5b D12b

Expression of individual P. falc. proteinsSDS Lysate

SDS LysateM

W, 0

.4ug

3C, 2

0ug

EQ

3255

B

EQ

3256

B

E

Q32

59B

EQ

3258

B

E

Q32

57B

EQ

3260

B

EQ

3261

B

EQ

3262

B

EQ

3263

B

EQ

3264

B

C1a/b C1a/b C2a/b C2a/b C6a/b C6a/b C8a/b C8a/b D3a/b D3a/b D5a/b D5a/b D12a/b D12a/b

EQ

3265

B

EQ

3267

B

EQ

3266

B

EQ

3268

B

Co-expression of P. falc. pairs

Co-expressed (large scale) and co-purified P. falc. pair E2/E3:

EQ3041B – Ubiquitin-conjugating enzyme E2

Ladd

er

E3 +

E2

E3 +

E2E3 E2

21

14

6

45

97116

31

66

E2 18.2 kDaE3 17.5 kDa

200

EQ3107B – Ubiquitin-protein ligase E3

ORF B ORF D

+

ORF BORF D

"FLIP"

ORF A

ORF B

ORF C

ORF D

ORF E

Goal: To co-express two proteins using a set of existing plasmids:

Set of expression plasmids:

Erin QuartleyChristina de VriesDaniela De RosaJulie BabulskiYoshiko KonSarah Mitchell

Elizabeth GrayhackEric PhizickyMark Dumont

University of Rochester:

Stanley FieldsWim Hol

Marissa VignaliDoug LaCountLori Schoenfeld

University of Washington (Seattle):