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 Screening: expression & solubility

Large-scale growth & purification Large-scale growth & purification

Large Small Large Small
proteins: proteins: proteins: proteins:
15
N label Se-Met 15
N label

Se-Met- C,15N-
13
C,15N-
13

label label label

X-ray NMR
 CESG’s wheat-germ cell-free protein expression project
represents a three-way collaboration over a 3-year period
 EhimeUniversity & Cell-Free Sciences Co. Ltd
(Matsuyama, Japan) (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 Production & analysis of 15N-protein
PCR from cDNA DNA plasmid preps
Ligation cloning Transcription
DNA plasmid preps Translation on [15N]-amino acids
  (4 ml reaction)
Small scale (50 l reaction) ts Isolation, purification (tag removal)
rge
Transcription HSQC NMR analysis
l ta

Translation Solubility, stability, & MS analysis

sfu
ce s

Analysis Production of [13C,15N]-protein

Suc

Expression level As above but with double-labeling

Solubility (4 – 12 ml reaction)
(Tag cleavage)
Screening Production
Structure for structural analysis
determination
 Small scale (50 l) results:
Arabidopsis ORFs with N-terminal (His)6 tags

Expression Solubility of (His)6-fusions

Yes 116 77 % High ( >50 %) 75 65 %

No 35 23 % Low (< 50 %) 41 35 %

Total 151 100 % Total 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 86 79% high ( >50 %) 56 65%

No 23 21%
low (< 50 %) 30 35%
Total 109 100%
Total 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 52 (both) 4
(His)6-fusion GST-fusion
after cleavage
 Large scale cell-free production for structural studies
N-(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)
1
N-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
 Wheat germ cell-free structure gallery

At3g01050 At2g24940
12 kDa 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

E. coli cells Wheat germ cell-free

Total tested 95 Total tested 93 90

All MBP-fusions Fusion: (His)6 GST
Expression – 39
Expression + 56 Expression – 11 11
(59 %) Expression + 82 79
(88 %) (88 %)
Insoluble 1
Soluble 55 Insoluble 30 35
(58 %) Cleavage – - 1
Cleavage + - 43
Soluble 52 44
 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,
Motoo Watanabe P50 GM 64598