markley
TRANSCRIPT
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Protein Production for
Structural InvestigationsBased on a Wheat Germ
Cell-Free Expression System
John L. Markley
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Where cell-free fits in the big picture of CESG
Please see the CESG posters
1. Pipeline overview
2. Constructs, E. colistrains and media (Terrific Broth
& chemically-defined), and expression screening
3. Large-scale E. colicell growth and protein
purification
4. Efficient labeling (Se-Met,
15
N, &
13
C;
15
N) of proteinsproduced from E. coli
5. High-throughput crystallomics
6. Cell-free protein production: expression screening &
production of labeled proteins
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Target selection
E. colicells
Screening: expression & solubility
Cell-free
Screening: expression & solubility
13C,15N-
label
Small
proteins:15N label
Large
proteins:
Se-Met
Large-scale growth & purification
Small
proteins:15N label
Large
proteins:
Large-scale growth & purification
13C,15N-
labelSe-Met-
label
NMRX-ray
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Target
Cloning Production & analysis of15N-protein
PCR from cDNA DNA plasmid preps
Ligation cloning TranscriptionDNA plasmid preps Translation on [15N]-amino acids
(4 ml reaction)
Small scale (50 Ql reaction) Isolation, purification (tag removal)
Transcription HSQC NMR analysis
Translation Solubility, stability, & MS analysis
Analysis Production of [13C,15N]-protein
Expression level As above but with double-labeling
Solubility (4 12 ml reaction)
(Tag cleavage)
Structure determination
Work-flow diagram: wheat germ cell-free approach for NMR
Screening Production for structural analysis
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Solubility of (His)6-fusions
High ( >50 %) 75 65 %
Low (< 50 %) 41 35 %
Total 116 100 %
Small scale (50 Ql) results:
Arabidopsis ORFs with N-terminal (His)6 tags
Overall success rate for producing highly soluble protein with
N-terminal (His)6tag: 50%
Expression
Yes 116 77 %
No 35 23 %
Total 151 100 %
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80 (both)
52 (both)
Expressed: 89 total
Soluble: 63 total
3 6
7 4
GST-fusion
GST-fusion
after cleavage
(His)6-fusion
(His)6-fusion
Screening results:
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Large scale cell-free production for structural studiesN-(His)6 tag N-GST tag
Number of proteins ([15N]-, 4 ml rxn): 49 3
Low yield (no HSQC) 26 (54 %) 0 (0 %)
Higher yield (HSQC possible) 23 (46 %) 3 (100 %)Average yield: 0.6 mg/ml 0.75 mg/ml
(following
cleavage)
1N-15N HSQC results:
+ (folded, non-aggregating) 14 (61 %) 1 (33 %)
- (unsuitable for NMR structure) 9 (39 %) 2 (67 %)
Number of proteins ([13C,15N]-, 4-12 ml rxn): 5 1
Structures solved 2 -
Structures in progress 3 1
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Robotics: CFS GeneDecoder 1000: delivered January 2004
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Two modes of operation for the GeneDecoder 1000
Screening
Uses 4 x 96-well plates
Overnight run Produces 2-10 Qg protein / well
Consumes 2.5 5 mL of wheat germ extract / plate
Small-scale protein production
Uses 2 x 96-well plates
Overnight run
Produces 10-50 Qg protein / well
Consumes 5 10 mL of wheat germ extract / plate
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Preliminary results from the Comparison Workgroup of96 Arabidopsis targets: (1) small-scale expression trials
E. colicells
Total tested 95All MBP-fusions
Expression 39
Expression + 56
(59 %)
Insoluble 1
Soluble 55
(58 %)
Wheat germ cell-free
Total tested 93 90Fusion: (His)6 GST
Expression 11 11
Expression + 82 79
(88 %) (88 %)
Insoluble 30 35
Cleavage - 1
Cleavage + - 43
Soluble 52 44
(56 %) (48 %)
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SummaryAdvantages
Cell-free method supports rapid and efficient screening
(supported by robotics)
Cell-free method requires smaller volumes (avoids lengthy
concentration steps in protein purification)
Labeled proteins can be prepared rapidly (in 1-2 days) to meetneeds of structural biologists
Supports labeling strategies that are not practical for proteins
produced from bacterial cells (no label scrambling)
Supports the production of eukaryotic N-terminal (His)6 proteins
(previous experience showed that these were not produced
successfully from E. colicells)
Disadvantages
Reagent intensive
Currently not compatible with Gateway cloning technology used
in other parts of the project
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32 kD CESG target: protein madein Tokyo by E. colicell-free; NMRstructure solved in Tokyo
Future cell-free plansRobotics
Automate the protein production (12 4-6 ml reactions / week)
Large-scale robot to be delivered by May 2004
Se-Met samples for X-ray crystallography
Successful for 3 proteins on a 50 Ql scale
Stereo array isotope labeling (SAIL) from wheat germ cell-free
Collaboration with M. Kainosho (Tokyo Metro. Univ.)
Complete the Comparison Workgroup96 targets produced from E. colicells and wheat germ cell free
E. colicells part complete (through 1H-15N HSQC of MBP-cleaved)
Cell-free screening nearly complete (His6- and GST-cleaved)
Cell-free 1H-15N HSQC in progress (His6- and GST-cleaved)
Targets from other eukaryotic genomes
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UW-Madison: Dave Aceti, Rick Amasino, Raj Arangarasan, Arash Bahrami, Craig
Bingman, Paul Blommel, Blake Buchan, Heather Burch, John Cao, Claudia
Cornilescu, Gabriel Cornilescu, Jurgen Doreleijers, Dave Dyer, Hamid Eghbalnia,
Brian Fox, Ronnie Fredrick, Holalkere Geetha, Premkum Gopalakrishnan, ByungWoo Han, Adrian Hegeman, Dave Hruby, Won Bae Jeon, Ken Johnson, Todd Kimball,
Kelly Kjer, John Kunert, Min S. Lee, Peter Lee, Jing Li, Scott Leisman, Miron Livny,
Andrew Markley, Zach Miller, Ramya Narayama, Craig Newman, George Phillips,
John Primm, Bryan Ramirez, Nitin Ravoof, Ivan Rayment, Megan Riters, Michael
Runnels, Kory Seder, Mark Shahan, Jeff Shaw, Shanteri Singh, David Smith, Jikui
Song, Hassan Sreenath, Mike Sussman, Sandy Thao, Ejan Tyler, Robert Tyer, Eldon
Ulrich, Dmitriy Vinarov, Frank Vojtik, Liya Wang, R. Kent Wenger, Gary Wesensberg,
Milo Westler, Russell Wrobel, Jianhua Zhang, Qin Zhao, Zsolt Zolnai
Medical College of Wisconsin: Betsy Lytle, Brian Volkman, Francis Peterson
Molecular Kinetics: Keith Dunker, Chris Oldfield
Hebrew University (Jerusalem): Michal Linial, Elon Portugaly, Ilone Kifer
German National Center for Health & Environment (Munich): Dmitrij Frishman
Tokyo Metropolitan University: Masatsune Kainosho, Yuko Katagiri, Nozomi
Sugimori, Akira M. Ono, Tsutomu Terauchi, Takuya Torizawa
Ehime University:Yaeta Endo, Tatsuya Sawasaki
CellFreeSciences, Inc. (Yokohama): Ryo Morishita, Mihoro Saeki,
Motoo Watanabe
CESG team members and collaborators
P50 GM 64598
Support