Post on 17-Jan-2016
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):