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pCOLD Vectors and Other
Alternative
Expression Systems
for Structural Genomics
Cold- shock adaptation of E. coli
37ºC
< 20 °C
Optimal Growth Acclimation
Phase
Steady Low Temp Growth
Adapted Cells
CSPs
Non-CSPs
Non-CSPs
CSPs
Stationary
Phase
Maps of pCold vectors
cspA 3’UTR
multiple cloning site
factor Xa site
His
TEE
6
cspA 5’UTR
lac operator
cspA 3’UTR
multiple cloning site
His
6
TEE
cspA 5’UTR
lac operator
cspA promoter
cspA promoter
pCold I
pCold II
(4.4 kb)
(4.4 kb)
cspA 3’UTR
multiple cloning site
cspA 3’UTR
multiple cloning site
cspA 5’UTR
TEE
cspA 5’UTR
lac operator
lac operator
cspA promoter
cspA promoter
pCold III
pCold IV
(4.4 kb)
(4.4 kb)
TEE (translation enhancing element): ATGAATCACAAAGTG (MNHKV)
His6: CATCATCATCATCATCAT
Factor Xa site: ATCGAAGGTAGG (IEGR)
Multiple cloning site:
NdeI
SacI
KpnI
XhoI
BamHI
EcoRI
HindIII
SalI
PstI
XbaI
CATATGGAGCTCGGTACCCTCGAGGGATCCGAATTCAAGCTTGTCGACCTGCAGTCTAGA
SDS-PAGE of whole cell lysates - E.coli EnvZ ATP-binding domain (EnvZ-B),
Xenopus calmodulin (CaM) and E.coli trigger factor expressed using pCold vectors
1 2 3 4 5
0 12 24 36 48 hr
1
T
2
S
 EnvZ-B
1 2 3 4
 EnvZ-B
5 6 7
 EnvZ-B
1
T
2
S
1
T
2
S
 trigger factor
 calmodulin
Expression level and solubility of different proteins - pColdI and pET14 systems
E.coli genes
gene
Human genes
pET14
pColdI
gene
expression solubility expression solubility
ER6
ER7
ER15
ER19
ER64
ER85
ER115
ER130
ER135
+
+++
+
+++
NE
+
++
+++
++
NS
NS
NS
+++
NA
NS
+++
+
+
+++
++
+++
+++
NE
+
++
++
++
NS
+
NS
+++
NA
NS
++
NS
+++
Drosophila genes
gene
HR8
HR31
HR520
HR521
HR522
HR524
HR529
HR535
HR540
pColdI
gene
++
+
NE
++
+++
+++
++
+
NE
++
++
NE
NE
+++
NE
+++
NS
NA
NS
NS
NA
NA
NS
NA
++
++
NE
+++
NE
NE
++
+++
NE
+++
+++
NA
NS
NA
NA
NS
NS
NA
C.elegans genes
pET14
++
NE
++
NE
pColdI
expression solubility expression solubility
expression solubility expression solubility
FR2
FR4
FR5
FR6
FR14
FR37
FR48
FR59
FR70
FR78
pET14
+
NA
+
NA
NS
++
NA
++
++
++
++
NE
+++
NE
++
+++
++
+++
+++
+++
++
NA
++
NA
NS
++
+++
++
++
++
pET14
pColdI
expression solubility expression solubility
WR13
WR24
WR26
WR27
WR33
WR35
WR41
WR44
WR49
WR53
+++
NE
+++
+++
+++
+++
+++
NE
+++
+++
NS
NA
NS
NS
+++
NS
++
NA
NS
+++
+++
NE
++
+++
+++
+++
++
+
+++
+++
NS
NA
NS
NS
+++
NS
+++
NS
NS
+++
In total, 38 genes as shown above were chosen and cloned in pColdI and pET14 vectors, respectively. Samples with better expression level and/or solubility in pET14
were labeled with blue color, and red color for those with better expression and/or solubility in pColdI. NS: not soluble; NE: no expression; NA: not available.
[1H-15N]-HSQC spectra of 15N-enriched Xenopus
calmodulin produced with pCold vector
Cell lysate NMR
Purified protein NMR
Sequential connectivity map summarizing the results of tripleresonance NMR experiments with calmodulin in whole cell lysates
~ 80% of the peaks are assigned
Target Selection, Cloning and Protein Production
Identify Target ORF
Human, C. elegans, Drosophila, Arabidopsis, yeast and others
Validate/ cDNA
Clone into Expression Vectors
N/C-Terminal His-Tags, pCold, No tag,
MBP, SUMO, Pichia, cell-free
Insoluble or
not expressed
Small Scale Expression
Soluble
Large Scale Fermentation/Protein Purification
Multiplex Expression System
Classical Restriction
Endonuclease/Ligasedependent cloning
E. coli Expression Vectors
Eukaryotic
Expression systems
Expanded multiplex system
Attempt to use fusion protein expression
systems in place of our standard T7 Multiplex
Expression System (Acton et al, submitted) for a set
of 50 eukaryotic target proteins
1) Gateway MBP-fusion expression system (Kapust & Waugh 1999)
(collaboration with D. Waugh, NCI)
2) SUMO system (Lifesensors, Inc. Malvern, PA)
(collaboration with T. Butt, Lifesensors, Inc.)
MBP fusion coupled with cleavage by TEV
MBP is cleaved from its fusion partner
by Tobacco Etch Virus (TEV) Proteinase
TEV recognizes the
consensus sequence:
Glu-X-X-Tyr-X-Gln-Ser
(Dougherty et al., 1989)
(Routzahn & Waugh 2002)
Both in vivo and in vitro
cleavage conditions
are being investigated
Interesting note: Recombinant TEV does not fold properly
in vivo and it must be generated as an MBP fusion as well
Summary of MBP screening
Target
pET
MBPfusion
MBPfusion
in vivo
cleavage
uncleaved
MBP-fusion in
vitro cleavage
Target
pET
MBPfusion
in vivo
cleavage
MBPfusion
uncleaved
MBP-fusion in
vitro cleavage
WR2
E/NS
NE
E/S
low
WR16
NE
NE
NE
N/A
WR3
E/NS
E/S
E/S
Y
WR18
PE
PE
E/S
low
WR4
E/S
E/S
E/S
Y
WR26
E/NS
NE
E/S
N/A
WR5
NE
NE
NE
N/A
WR27
E/NS
E/NS
E/S
low
WR6
NE
NE
E/S
Y
WR28
NE
NE
NE
N/A
WR8
E/S
NE
E/S
Y
WR39
E/S
E/S
E/S
low
WR9
NE
NE
NE
N/A
WR41
E/S
E/S
E/S
Y
WR10
NE
NE
E/S
low
WR43
E/S
E/S
E/S
N/A
WR11
E/S
E/S
E/S
Y
WR44
NE
NE
NE
N/A
WR13
E/NS
E/NS
E/S
N/A
WR49
E/NS
E/S
E/S
low
WR14
E/S
E/S
E/S
Y
WR53
E/S
E/S
E/S
Y
WR15
NE
NE
NE
N/A
WR54
E/NS
NE
NE
N/A
Not expressed
Expressed/soluble
Expressed/insoluble
SUMO system
6хHis
SUMO
Protein of interest
SUMP protease (Ulp1)
• SUMO is a Ubiquitin-like (UBL) protein
• UBLs are small, highly soluble, globular proteins
• SUMO has been reported to exhibit chaperone-like activity
• Ulp1 is used to cleave the protein target from
the fusion by recognizing the entire SUMO protein
• SUMO system utilizes Ni-affinity chromatography
• Cleavage must occur in vitro
(Figure courtesy of www.lifesensors.com)
Summary of SUMO fusion screening
pET
(N-terminal His-tag)
SUMO fusion
AR5
NE
NE
AR22
E/S
E/S
FR10
E/NS
E/S
HR894
E/NS
E/NS
HR1553
E/NS
E/S
HR1686
E/NS
E/NS
HR1697
NE
E/S
E/NS
E/NS
Target ID
WR26
Not expressed
Expressed/soluble
Expressed/insoluble
A stable cell-free protein synthesis
system prepared from wheat germ
CK
DHFR
Improvement in preparing cell
extracts: removing endosperm
contaminants, including tritin, thionin,
ribonucleases, deoxy ribonucleases,
and proteases, by thorough washing
and sonication.
(Proc. Natl. Acad. Sci. USA 99, 14652-57)
(collaboration with Y. Endo, Ehime University)
Protein synthesis in the dialysis system. (A and B) Coomassie blue-stained
SDS polyacrylamide gels showing DHFR synthesis with (A) or without (B)
addition of new mRNA. Arrows and asterisks mark DHFR and creatine kinase,
respectively. (C) Amounts of DHFR synthesized as determined from
densitometric scans of the gels in A (closed circles) and B (open circles).
A cell-free expression vector and its performance
GFP
mRNA supplement (92 mg in 500 ml reaction each time)
(a) Schematic illustration of pEU. (b) SDS/PAGE analysis of GFP produced during 14 days of reaction. mRNA produced by transcription of
circular pEU was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1-µl aliquot of the mixture
was run on the gel, and protein bands were stained with CBB. The arrow shows GFP; "st" designates an authentic GFP band (0.5 µg).
Protein synthesis of human targets by
wheat-germ system
*
*
*
*
*
*
*
*
*
Summary of screening by cell-free system from wheat germ
pET(N-terminal tag)
Target ID
Not expressed
Expressed/soluble
Expressed/insoluble
AR15
AR16
AR21
AR22
FR10
HR812
HR894
HR919
HR945
HR969
HR1553
HR1576
HR1686
HR1697
HR1719
HR1722
HR1738
WR13
WR19
WR20
WR21
WR23
WR24
WR26
WR27
WR35
WR38
WR41
WR43
WR44
WR50
WR53
WR54
Protein synthesis by wheatgerm system
Expression
Solubility
Expression
Solubility
+++
NE
NE
+
+++
NE
+++
+++
+++
+++
+++
0.5
+++
NE
+++
+++
+++
+++
+++
NE
+++
+++
NE
+++
+++
+++
++
+++
+
NE
+
+++
+++
+
NA
NA
+++
NS
NA
NS
NS
NS
++
NS
NS
NS
NA
NS
NS
NS
NS
NS
NA
+
NS
NA
NS
NS
NS
NS
+++
++
NA
NS
+++
NS
++
++
+
+++
+
+++
NE
++
NE
++
NE
NE
++
NE
+
+
NE
+++
+++
+++
0.5
+++
NE
+++
+++
+++
+++
+++
+
+++
++
++
++
++
++
+++
0.5
+++
++
NA
+++
NA
+++
NA
NA
++
NA
+++
+
NA
0.5
0.5
NS
NS
NS
NA
NS
0.5
+++
0.5
+++
+
NS
++
+++
+++
Panel of 50 eukaryotic targets in eight expression systems
Eukaryotic Core 50E. coli pET (Nter-H6)
Number
Target ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
AR5
AR15
AR16
AR21
AR22
FR4
FR10
FR59
HR79
HR91
HR547
HR812
HR894
HR919
HR945
HR969
HR1553
HR1576
HR1686
HR1697
HR1719
HR1722
HR1738
HR1854
HR1869
HR1889
HR1913
HR1953
WR12
WR13
WR19
WR20
WR21
WR23
WR24
WR26
WR27
WR28
WR35
WR38
WR40
WR41
WR42
WR43
WR44
WR49
WR50
WR51
WR53
WR54
H
E
S
E. coli pET (Cter-H6)
H
E
NA
H
E
S
NA
NA
NA
NA
NA
NA
NA
E. coli MBP + TEV
H
E
S
E. coli MBP (uncut)
H
E
S
E. coli SUMO fusions
H
E
S
Wheat germ extract
H
E
S
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
LEGEND:
=
=
=
=
=
=
=
=
=
S
E. coli pCOLD vector
not attempted yet
hook up complete
H = hooked up
expressed
E = expression tested
not expressed
S = solubility
insoluble
soluble
not applicable
in progress
expressed & soluble in at least one system
P. pastoris pPIC3.5
H
E
S
Acknowledgements
Guoliang Qing
Li-Chung Ma
Ahmad Khorchid
G. V. T. Swapna
Tapas K. Mal
Masanori Mitta Takayama
Bing Xia
Sangita Phadtare
Haiping Ke
Gaetano T. Montelione
Mitsuhiko Ikura
Masayori Inouye
Thomas Acton
Rong Xiao
Ritu Shastry
Chi Kent Ho
Natalia Denissova
Bonnie Cooper
Kellie Cunningham
Liang-yu (Lydia) Shih
Yi-Wen Chiang
Shin-Geon Choi
Tatsuya Sawasaki
Yaeta Endo
Kate Drahos
Tauseef R. Butt
David S. Waugh
What if…?
PCR & cloning of ORFs
Unexpressed
or Insoluble
Proteins
Construct validation
Small-scale expression
& solubility screens
Protein Production Pipeline
PCR & cloning of ORFs
Analysis & passing of
targets to fermentation
Large-scale production &
purification of 15N/13C, or
selenomethionine-labeled
proteins
Structure determination
by X-ray crystallography
Construct validation
Small-scale expression
& solubility screens
using T7 system
Structure determination
by NMR