1. Protein Production for Structural Investigations Based on a Wheat Germ Cell-Free Expression System John L. Markley markley@nmrfam.wisc.edu

2. 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, 15 N, & 13 C; 15 N) of proteins produced from E. coli 5.High-throughput crystallomics 6.Cell-free protein production: expression screening & production of labeled proteins

3. Target selection E. coli cells Screening: expression & solubility Cell-free Screening: expression & solubility 13 C, 15 N- label Small proteins: 15 N label Large proteins: Se-Met Large-scale growth & purification Small proteins: 15 N label Large proteins: Large-scale growth & purification 13 C, 15 N- label Se-Met- label NMR X-ray

4. Ehime University & 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 CESG’s wheat-germ cell-free protein expression project represents a three-way collaboration over a 3-year period

5. Target Cloning Production & analysis of 15 N-protein PCR from cDNA DNA plasmid preps Ligation cloning Transcription DNA plasmid preps Translation on [ 15 N]-amino acids (4 ml reaction) Small scale (50  l reaction) Isolation, purification (tag removal) Transcription HSQC NMR analysis Translation Solubility, stability, & MS analysis Analysis Production of [ 13 C, 15 N]-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 Successful targets

6. Solubility of (His) 6 -fusions High ( >50 %) 75 65 % Low (< 50 %) 41 35 % Total 116 100 % Small scale (50  l) results: Arabidopsis ORFs with N-terminal (His) 6 tags Overall success rate for producing highly soluble protein with N-terminal (His) 6 tag: 50% Expression Yes 116 77 % No 35 23 % Total 151 100 %

7. 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% Expression Yes 86 79% No 23 21% Total 109 100% Solubility of GST fusions high ( >50 %) 56 65% low (< 50 %) 30 35% Total 86 100% Small scale (50  l) results: Arabidopsis ORFs with N-terminal GST tags

8. 80 (both) 52 (both) Expressed: 89 total Soluble: 63 total 36 74 GST-fusion GST-fusion after cleavage (His) 6 -fusion Screening results:

9. Large scale cell-free production for structural studies N-(His) 6 tag N-GST tag Number of proteins ([ 15 N]-, 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- 15 N HSQC results: + (folded, non-aggregating) 14 (61 %) 1 (33 %) - (unsuitable for NMR structure) 9 (39 %) 2 (67 %) Number of proteins ([ 13 C, 15 N]-, 4-12 ml rxn): 5 1 Structures solved 2 - Structures in progress 3 1

10. At3g01050 12 kDa At2g24940 14 kDa Wheat germ cell-free structure gallery

11. Robotics: CFS GeneDecoder 1000: delivered January 2004

12. 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

13. Preliminary results from the ‘Comparison Workgroup’ of 96 Arabidopsis targets: (1) small-scale expression trials E. coli cells Total tested 95 All MBP-fusions Expression – 39 Expression + 56 (59 %) Insoluble 1 Soluble 55 (58 %) Wheat germ cell-free Total tested 93 90 Fusion: (His) 6 GST Expression – 11 11 Expression + 82 79 (88 %) (88 %) Insoluble 30 35 Cleavage – - 1 Cleavage + - 43 Soluble 52 44 (56 %) (48 %)

14. 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

15. 32 kD CESG target: protein made in Tokyo by E. coli cell-free; NMR structure solved in Tokyo 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.) Complete the ‘Comparison Workgroup’ 96 targets produced from E. coli cells and wheat germ cell free E. coli cells part complete (through 1 H- 15 N HSQC of MBP-cleaved) Cell-free screening nearly complete (His 6 - and GST-cleaved) Cell-free 1 H- 15 N HSQC in progress (His 6 - and GST-cleaved) Targets from other eukaryotic genomes

16. 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 Ehime University: Yaeta Endo, Tatsuya Sawasaki CellFreeSciences, Inc. (Yokohama): Ryo Morishita, Mihoro Saeki, Motoo Watanabe CESG team members and collaborators P50 GM 64598 Support