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:

1

2

3

4

1 2

34

1 anneals to 4 only

2 anneals to 3 only

ORF A ORF B

ORF B

ORF A

1

2

3

4

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 B

ORF 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 2

insert 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

97

116

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

10

12

4

6

8

1012

E3

+ E

2

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

, 20

ug

EQ

29

95B

EQ

29

97B

EQ

30

05B

EQ

30

09B

E

Q3

041

B

EQ

30

27B

E

Q3

023

B

EQ

30

43B

EQ

30

47B

EQ

30

61B

EQ

30

63B

EQ

30

71B

EQ

30

75B

EQ

30

93B

EQ

30

89B

EQ

31

07B

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

Expression of individual P. falc. proteins

SDS Lysate

SDS LysateM

W, 0

.4u

g

3C

, 20

ug

EQ

32

55B

EQ

32

56B

E

Q3

259

B

EQ

32

58B

E

Q3

257

B

EQ

32

60B

EQ

32

61B

EQ

32

62B

EQ

32

63B

EQ

32

64B

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

32

65B

EQ

32

67B

EQ

32

66B

EQ

32

68B

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

+ E

2

E3

+ E

2E3 E2

21

14

6

45

97

116

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):