pcold vectors and other alternative expression systems for structural genomics
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Optimal Growth Acclimation Steady Low Temp Growth Stationary Phase Adapted Cells Phase
Non-CSPs
Non-CSPs
CSPs
CSPs
Growth Curve
Cold- shock adaptation of E. coli
< 20 °C37ºC
pCold I (4.4 kb)
lacI
ColE1 ori
Am
p
M13 IG
cspA promoterlac operatorcspA 5’UTRTEEHis6
multiple cloning sitecspA 3’UTR
factor Xa site
TEE (translation enhancing element): ATGAATCACAAAGTG (MNHKV)His6: CATCATCATCATCATCATFactor Xa site: ATCGAAGGTAGG (IEGR)Multiple cloning site:
CATATGGAGCTCGGTACCCTCGAGGGATCCGAATTCAAGCTTGTCGACCTGCAGTCTAGA
NdeI SacI KpnI XhoI BamHI EcoRI HindIII SalI PstI XbaI
Maps of pCold vectors
pCold III (4.4 kb) la
cI
ColE1 ori
Am
p
M13 IGlac operatorcspA 5’UTRTEE
multiple cloning sitecspA 3’UTR
cspA promoter
pCold IV (4.4 kb) la
cI
ColE1 ori
Am
p
M13 IGlac operator
cspA 5’UTRmultiple cloning sitecspA 3’UTR
cspA promoter
pCold II (4.4 kb)
M13 IG
Am
p
ColE1 ori
lacI
lac operatorcspA 5’UTRTEE
His6
multiple cloning sitecspA 3’UTR
cspA promoter
T S1 2
trigger factor
EnvZ-B
T S1 2
T S
calmodulin
1 2
1 2 3 4 5 0 12 24 36 48 hr
EnvZ-B
1 2 3 4 5 6 7
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
EnvZ-B
Expression level and solubility of different proteins - pColdI and pET14 systems
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.
Human genes
Drosophila genes
pET14 pColdIgene
expression solubility expression solubility
FR2FR4FR5FR6FR14FR37FR48FR59FR70
++NE++
+++
NE+++++
+NA+
NS
++NA
+++
++
+++NE
++ NS
++ +++++
+++++++
++ +++ ++
++
NA++
FR78
NE NA NE NA
+++ ++ +++ ++
C.elegans genes
E.coli genes
pET14 pColdIgene
expression solubility expression solubility
ER6ER7ER15ER19ER64ER85ER115ER130ER135
++++
++++NE+
+++++++
NSNSNS+++NA
NS+++
+
+++++
+++NE NA
NS++++
++NS+
+ ++ +++
NS+
NS+++
+++
pColdIpET14gene
expression solubility expression solubility
HR8HR31HR520HR521HR522HR524HR529HR535HR540
+++
NE++
+++
+++NSNANS
NA NE
++
NE++
NS
NA
NSNA
+++NA
++++++NA
++ NS NA
NE NA NS
NE
NS
+++
NENE
++
NE
pET14 pColdIgene
expression solubility expression solubility
WR13WR24WR26WR27WR33WR35WR41WR44WR49
+++NE+++++++++++++++NE+++
NSNANSNS+++
NS++
+++
+++
++NE
++++++ +++
NS+++
+++NSNA
NS +++ NS
NS
NANSNS
WR53 +++ +++ +++ +++
Purified protein NMRCell lysate NMR
[1H-15N]-HSQC spectra of 15N-enriched Xenopus calmodulin produced with pCold vector
Sequential connectivity map summarizing the results of triple-
resonance NMR experiments with calmodulin in whole cell lysates
~ 80% of the peaks are assigned
Target Selection, Cloning and Protein Production
Validate/ cDNA
Identify Target ORF
Clone into Expression Vectors
Human, C. elegans, Drosophila, Arabidopsis, yeast and others
N/C-Terminal His-Tags, pCold, No tag, MBP, SUMO, Pichia, cell-free
Small Scale Expression
Large Scale Fermentation/Protein Purification
Soluble
Insoluble ornot expressed
Multiplex Expression System
Classical Restriction Endonuclease/Ligase-
dependent cloning
E. coli Expression Vectors
Eukaryotic Expression systems
Expanded multiplex system
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.)
Attempt to use fusion protein expression systems in place of our standard T7 Multiplex Expression System (Acton et al, submitted) for a setof 50 eukaryotic target proteins
MBP fusion coupled with cleavage by TEV
MBP is cleaved from its fusion partnerby Tobacco Etch Virus (TEV) Proteinase
TEV recognizes the consensus sequence:
Glu-X-X-Tyr-X-Gln-Ser
(Routzahn & Waugh 2002)
Both in vivo and in vitro cleavage conditionsare being investigated
Interesting note: Recombinant TEV does not fold properlyin vivo and it must be generated as an MBP fusion as well
(Dougherty et al., 1989)
Summary of MBP screening
N/ANENENEWR15
Y E/SE/SE/SWR14
N/AE/SE/NSE/NSWR13
Y E/SE/SE/SWR11
lowE/SNENEWR10
N/ANENENEWR9
Y E/SNEE/SWR8
Y E/SNENEWR6
N/ANENENEWR5
Y E/SE/SE/SWR4
YE/SE/SE/NSWR3
lowE/SNEE/NSWR2
MBP-fusion in vitro cleavage
MBP-fusion
uncleaved
MBP-fusion in vivo
cleavage
pETTarget
Not expressed
Expressed/soluble
Expressed/insoluble
N/ANENEE/NSWR54
Y E/SE/SE/SWR53
lowE/SE/SE/NSWR49
N/ANENENEWR44
N/AE/SE/SE/SWR43
YE/SE/SE/SWR41
lowE/SE/SE/SWR39
N/ANENENEWR28
lowE/SE/NSE/NSWR27
N/AE/SNEE/NSWR26
lowE/SPEPEWR18
N/ANENENEWR16
MBP-fusion in vitro cleavage
MBP-fusion
uncleaved
MBP-fusion in vivo
cleavage
pETTarget
SUMO system
• 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)
6хHis SUMO Protein of interest
SUMP protease (Ulp1)
Summary of SUMO fusion screening
Target IDpET
(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
WR26 E/NS E/NS
Not expressed
Expressed/soluble
Expressed/insoluble
A stable cell-free protein synthesis system prepared from wheat germ
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)
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).
DHFR
CK
(collaboration with Y. Endo, Ehime University)
A cell-free expression vector and its performance
GFP
mRNA supplement (92 g in 500 l 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).
Target ID
pET(N - terminal tag) Protein synthesis by wheat -
germ system
Expression Solubility Expression Solubility
AR15 +++ + ++ ++ AR16 NE NA ++ ++
AR21 NE NA + +++ AR22 + +++ +++ 0.5 FR10 +++ NS + +++ HR812 NE NA +++ ++ HR894 +++ NS NE NA HR919 +++ NS ++ +++ HR945 +++ NS NE NA HR969 +++ ++ ++ +++ HR1553 +++ NS NE NA HR1576 0.5 NS NE NA HR1686 +++ NS ++ ++ HR1697 NE NA NE NA HR1719 +++ NS + +++ HR1722 +++ NS + + HR1738 +++ NS NE NA WR13 +++ NS +++ 0.5 WR19 +++ NS +++ 0.5 WR20 NE NA +++ NS WR21 +++ + 0.5 NS WR23 +++ NS +++ NS WR24 NE NA NE NA WR26 +++ NS +++ NS WR27 +++ NS +++ 0.5 WR35 +++ NS +++ +++ WR38 ++ NS +++ 0.5 WR41 +++ +++ +++ +++ WR43 + ++ + + WR44 NE NA +++ NS WR50 + NS ++ ++ WR53 +++ +++ ++ +++ WR54 +++ NS ++ +++
Summary of screening by cell-free system from wheat germ
Not expressed
Expressed/soluble
Expressed/insoluble
1 AR5 NA NA NA NA2 AR153 AR16 NA NA NA4 AR21 NA5 AR22 NA6 FR4 NA NA7 FR108 FR599 HR79 NA10 HR9111 HR547 NA NA12 HR812 NA NA NA13 HR894 NA NA14 HR91915 HR945 NA NA16 HR96917 HR1553 NA18 HR1576 NA NA19 HR1686 NA20 HR1697 NA NA NA21 HR171922 HR1722 NA23 HR1738 NA NA24 HR185425 HR1869 NA26 HR1889 NA27 HR191328 HR1953 NA NA29 WR12 NA30 WR1331 WR1932 WR20 NA33 WR2134 WR2335 WR24 NA NA NA NA36 WR26 NA NA NA37 WR2738 WR28 NA NA39 WR35 NA40 WR3841 WR40 NA NA NA42 WR4143 WR42 NA NA NA44 WR4345 WR44 NA NA NA NA46 WR4947 WR50 NA48 WR51 NA49 WR5350 WR54 NA NA
LEGEND:
= not attempted yet = hook up complete H = hooked up = expressed E = expression tested = not expressed S = solubility = insoluble = soluble
NA = not applicable = in progress = expressed & soluble in at least one system
Wheat germ extract
H E SSS
E. coli pET (Cter-H 6)
H E
E. coli pCOLD vector
H E SNumber Target ID H
Eukaryotic Core 50
E S
E. coli pET (Nter-H 6) E. coli MBP (uncut)
H E S
E. coli MBP + TEV
H E
E. coli SUMO fusions
H E S
P. pastoris pPIC3.5
H E S
Panel of 50 eukaryotic targets in eight expression systems
Acknowledgements
Guoliang Qing Thomas Acton Tatsuya SawasakiLi-Chung Ma Rong Xiao Yaeta Endo Ahmad Khorchid Ritu Shastry Kate Drahos G. V. T. Swapna Chi Kent Ho Tauseef R. ButtTapas K. Mal Natalia Denissova David S. WaughMasanori Mitta Takayama Bonnie CooperBing Xia Kellie CunninghamSangita Phadtare Liang-yu (Lydia) ShihHaiping Ke Yi-Wen Chiang Gaetano T. Montelione Shin-Geon ChoiMitsuhiko Ikura Masayori Inouye
What if…?
PCR & cloning of ORFs Construct validation
Small-scale expression & solubility screens
Unexpressed or Insoluble
Proteins
Protein Production Pipeline
PCR & cloning of ORFs Construct validation
Small-scale expression & solubility screens
using T7 system
Analysis & passing oftargets to fermentation
Large-scale production &purification of 15N/13C, or selenomethionine-labeled proteins
Structure determination by NMR
Structure determinationby X-ray crystallography
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