Supplementary Material
Expression Platforms for Producing Eukaryotic Proteins: A Comparison of E. coli Cell-Based and Wheat
Germ Cell-Free Synthesis, Affinity and Solubility Tags, and Cloning Strategies
David J. Aceti, Craig A. Bingman, Russell L. Wrobel, Ronnie O. Frederick, Shin-ichi Makino, Karl W. Nichols,
Sarata C. Sahu, Lai F. Bergeman, Paul G. Blommel, Claudia C. Cornilescu, Katarzyna A. Gromek, Kory D.
Seder, Soyoon Hwang, John G. Primm, Grzegorz Sabat, Frank C. Vojtik, Brian F. Volkman, Zsolt Zolnai,
George N. Phillips, Jr., John L. Markley, and Brian G. Fox.
---------------------------D. J. Aceti • C. A. Bingman • R. L. Wrobel • R. O. Frederick • S. Makino • K. W. Nichols • S. C. Sahu • L. F.
Bergeman • P. G. Blommel • C. C. Cornilescu • K. A. Gromek • K. D. Seder • S. Hwang • J. G. Primm • G.
Sabat • F. C. Vojtik • Z. Zolnai • G. N. Phillips, Jr. • J. L. Markley • B. G. Fox
The Center for Eukaryotic Structural Genomics, Nuclear Magnetic Resonance Facility at Madison, Department
of Biochemistry, University of Wisconsin at Madison, 433 Babcock Dr., Madison, Wisconsin 53706
B. F. Volkman
The Center for Eukaryotic Structural Genomics, Department of Biochemistry, Medical College of Wisconsin,
8701 W. Watertown Plank Rd., Milwaukee, WI 53226
S. C. Sahu • C. C. Cornilescu • J. L. Markley
Nuclear Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin at
Madison, 433 Babcock Dr., Madison, Wisconsin 53706
C. A. Bingman • G. N. Phillips, Jr.
Natural Product Biosynthesis, BioSciences at Rice and Department of Chemistry, Rice University, Houston, TX
77251
D. J. Aceti • C. A. Bingman • R. L. Wrobel • R. O. Frederick • S. Makino • K. A. Gromek • J. G. Primm • J. L.
Markley
Mitochondrial Protein Partnership, Department of Biochemistry, University of Wisconsin at Madison, 433
Babcock Dr., Madison, Wisconsin 53706
D. J. Aceti • C. A. Bingman • R. L. Wrobel • R. O. Frederick • S. Makino • K. A. Gromek • J. G. Primm • B. G.
Fox.
Transmembrane Protein Center, Department of Biochemistry, University of Wisconsin at Madison, 433
Babcock Dr., Madison, Wisconsin 53706
G. N. Phillips, Jr.
Current affiliation: BioSciences at Rice and Department of Chemistry, Rice University, Houston, TX 77251
B. G. Fox
Corresponding author: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr.,
Madison, Wisconsin 53706, Tel:(608) 262-9708, E-mail: bgfox@wisc.edu, FAX: 608-262-4693
1
Online Resource 1 contains the sources of clones used to construct the control workgroup and catalytic assays
or other characteristic biophysical measurement used with the expressed proteins.
2
Online Resource 1 Sources of clones used and catalytic assays where available
#
ORF ID
Protein Name
cDNA source
1
GO.34382
cytoplasmic dynein light chain
MGC:37100a
2
GO.6042
unknown protein At1g77540.1
Plant tissue cultureb
3
GO.14751
thioredoxin h1
Plant tissue culture
4
GO.33810
zinc finger protein
MGC:3359
5
GO.81370
unknown heme-binding protein
Plant tissue culture
6
GO.2361
unknown protein At1g01470.1
Plant tissue culture
7
GO.11624
thioredoxin-like protein
Plant tissue culture
8
GO.91592
allene oxide cyclase variant 1
Plant tissue culture
9
GO.91593
allene oxide cyclase variant 2
Plant tissue culture
10 GO.605
phosphatase
11 GO.91571
Catalytic assay
Reference
Spectrophotometric assay of NADPH/insulin-disulfide redox
reaction
[1]
Red-colored from heme binding
[2]
Plant tissue culture
pNPP phosphatase assay
[3]
enhanced C3 green fluorescent protein
Synthesizedc
Fluorescence spectrum
[4]
12 GO.91591
TEV protease
gift
Fluoresence anisotropy-based protease assay
[5]
13 GO.80048
sarcosine dimethylglycine methyltransferase
Plant tissue culture
Reaction with S-Adenosylmethionine followed by TLC
[6]
14 GO.37540
glyoxylate/hydroxypyruvate reductase
MGC:3078
NADPH-linked spectrophotometric assay
[7]
15 GO.79368
aspartoacylase
MGC:34517
Coupled assay following deamination of Asp
[8]
16 GO.70653
dimetal phosphatase
MGC:64213
pNPP phosphatase assay
[3]
17 GO.7312
putative steroid sulfotransferase
Plant tissue culture
18 GO.8210
12-oxophytodienoate-10,11-reductase
Plant tissue culture
Yellow-colored from FMN binding, NADPH-dependent
reduction of TNT
[9, 10]
19 GO.24674
agmatine iminohydrolase
Plant tissue culture
Assay of ammonia product by Nessler’s reagent
[11]
20 GO.34351
unknown protein
MGC:86070
21 GO.74329
Photinus (firefly) luciferase
Promega
Luciferase assay
[12]
a
cDNA clones from MGC, the Mammalian Gene Collection, are available from Open Biosystems, Huntsville, AL. b cDNA library prepared from
total mRNA obtained from Arabidopsis thaliana T87 tissue culture. c A variant GFP gene with fluorescence-enhancing mutations.
3
References
1. Holmgren A (1979) Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem 254(19):96279632
2. Tsutsui K, Mueller GC (1982) A protein with multiple heme-binding sites from rabbit serum. J Biol Chem 257(7):3925-3931
3. Kuznetsova E, Proudfoot M, Sanders SA, Reinking J, Savchenko A, Arrowsmith CH, Edwards AM, Yakunin AF (2005) Enzyme genomics:
Application of general enzymatic screens to discover new enzymes. FEMS Microbiol Rev 29(2):263-279
4. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science
263(5148):802-805
5. Blommel PG, Fox BG (2007) A combined approach to improving large-scale production of tobacco etch virus protease. Protein expression and
purification 55(1):53-68
6. McCoy JG, Bailey LJ, Ng YH, Bingman CA, Wrobel R, Weber AP, Fox BG, Phillips GN, Jr. (2009) Discovery of sarcosine dimethylglycine
methyltransferase from Galdieria sulphuraria. Proteins 74(2):368-377
7. Tolbert NE, Yamazaki RK, Oeser A (1970) Localization and properties of hydroxypyruvate and glyoxylate reductases in spinach leaf particles. J
Biol Chem 245(19):5129-5136
8. Kaul R, Casanova J, Johnson AB, Tang P, Matalon R (1991) Purification, characterization, and localization of aspartoacylase from bovine brain.
Journal of neurochemistry 56(1):129-135
9. Blehert DS, Fox BG, Chambliss GH (1999) Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases. Journal of
bacteriology 181(20):6254-6263
10. Schaller F, Weiler EW (1997) Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid
signaling pathway from Arabidopsis thaliana. Structural and functional relationship to yeast old yellow enzyme. J Biol Chem 272(44):2806628072
11. Yanagisawa H, Suzuki Y (1981) Corn Agmatine Iminohydrolase: PURIFICATION AND PROPERTIES. Plant physiology 67(4):697-700
12. Holmsen H, Holmsen I, Bernhardsen A (1966) Microdetermination of adenosine diphosphate and adenosine triphosphate in plasma with firefly
luciferase system. Analytical biochemistry 17(3):456-473
4