Protein Production for
Structural Investigations
Based on a Wheat Germ
Cell-Free Expression System
John L. Markley
markley@nmrfam.wisc.edu
Where cell-free fits in the big picture of CESG
Please see the CESG posters
1. Pipeline overview
2. Constructs, E. coli strains and media (Terrific Broth
& chemically-defined), and expression screening
3. Large-scale E. coli cell growth and protein
purification
4. Efficient labeling (Se-Met, 15N, & 13C;15N) of proteins
produced from E. coli
5. High-throughput crystallomics
6. Cell-free protein production: expression screening &
production of labeled proteins
Target selection
E. coli cells
Cell-free
Screening: expression & solubility
Large-scale growth & purification
Large
proteins:
Small
proteins:
15N label
Se-Metlabel
X-ray
Screening: expression & solubility
Large-scale growth & purification
Large
proteins:
Se-Met
Small
proteins:
15N label
13C,15N-
13C,15N-
label
label
NMR
CESG’s wheat-germ cell-free protein expression project
represents a three-way collaboration over a 3-year period
Ehime University
(Matsuyama, Japan)
&
Cell-Free Sciences Co. Ltd
(Yokohama, Japan)
Wheat - germ extract
Enabling methodology
Robotics
CESG
Screening of potential targets from eukaryotic genomes for suitability
for structural studies
Production of labeled proteins on the scale of several milligrams
Assessment of this approach for high-throughput structure
determination
Improvement of the technology through its use in a production
environment
Work-flow diagram: wheat germ cell-free approach for NMR
Target
Cloning
PCR from cDNA
Ligation cloning
DNA plasmid preps
Small scale (50 l reaction)
Transcription
Translation
Analysis
Expression level
Solubility
(Tag cleavage)
Screening
Production & analysis of 15N-protein
DNA plasmid preps
Transcription
Translation on [15N]-amino acids
(4 ml reaction)
Isolation, purification (tag removal)
HSQC NMR analysis
Solubility, stability, & MS analysis
Production of [13C,15N]-protein
As above but with double-labeling
(4 – 12 ml reaction)
Structure determination
Production for structural analysis
Small scale (50 l) results:
Arabidopsis ORFs with N-terminal (His)6 tags
Expression
Solubility of (His)6-fusions
Yes
No
116
35
77 %
23 %
Total
151 100 %
High ( >50 %)
Low (< 50 %)
Total
75
41
65 %
35 %
116 100 %
Overall success rate for producing highly soluble protein with
N-terminal (His)6 tag: 50%
Small scale (50 l) results:
Arabidopsis ORFs with N-terminal GST tags
Expression
Solubility of GST fusions
Yes
No
86
23
79%
21%
Total
109
100%
high ( >50 %)
low (< 50 %)
Total
56
30
65%
35%
86
100%
All 56 soluble fusion proteins were cleaved at PreScission™
protease sites: of these 55 remained soluble (>98%)
Overall success rate for producing highly soluble protein
following cleavage of GST fusions: 49%
Screening results:
Expressed: 89 total
3
80 (both)
6
(His)6-fusion
GST-fusion
Soluble: 63 total
7
(His)6-fusion
52 (both)
4
GST-fusion
after cleavage
Large scale cell-free production for structural studies
N-(His)6 tag
Number of proteins ([15N]-, 4 ml rxn):
Low yield (no HSQC)
Higher yield (HSQC possible)
Average yield:
N-GST tag
49
3
26 (54 %)
23 (46 %)
0.6 mg/ml
0 (0 %)
3 (100 %)
0.75 mg/ml
(following
cleavage)
14 (61 %)
9 (39 %)
1 (33 %)
2 (67 %)
1N-15N
HSQC results:
+ (folded, non-aggregating)
- (unsuitable for NMR structure)
Number of proteins ([13C,15N]-, 4-12 ml rxn):
Structures solved
Structures in progress
5
2
3
1
1
Wheat germ cell-free structure gallery
At3g01050
12 kDa
At2g24940
14 kDa
Robotics: CFS GeneDecoder 1000: delivered January 2004
Two modes of operation for the GeneDecoder 1000
Screening
•
•
•
•
Uses 4 x 96-well plates
Overnight run
Produces 2-10 g 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 g protein / well
Consumes 5 – 10 mL of wheat germ extract / plate
Preliminary results from the ‘Comparison Workgroup’ of
96 Arabidopsis targets: (1) small-scale expression trials
Wheat germ cell-free
E. coli cells
Total tested
All MBP-fusions
Expression –
Expression +
Insoluble
Soluble
95
39
56
(59 %)
1
55
(58 %)
Total tested
Fusion:
Expression –
Expression +
Insoluble
Cleavage –
Cleavage +
Soluble
93
(His)6
11
82
(88 %)
30
52
(56 %)
90
GST
11
79
(88 %)
35
1
43
44
(48 %)
Summary
Advantages
• 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 meet
needs 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. coli cells)
Disadvantages
• Reagent intensive
• Currently not compatible with Gateway cloning technology used
in other parts of the project
Future cell-free plans
Robotics
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 l scale
Stereo array isotope labeling (SAIL) from wheat germ cell-free
• Collaboration with M. Kainosho (Tokyo Metro. Univ.)
32 kD CESG target: protein made
in Tokyo by E. coli cell-free; NMR
structure solved in Tokyo
Complete the ‘Comparison Workgroup’
96 targets produced from E. coli cells and wheat germ cell free
• E. coli cells 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
CESG team members and collaborators
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, Byung
Woo 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
Support
Ehime University: Yaeta Endo, Tatsuya Sawasaki
CellFreeSciences, Inc. (Yokohama): Ryo Morishita, Mihoro Saeki,
P50 GM 64598
Motoo Watanabe