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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:. - PowerPoint PPT Presentation
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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):