A “Control Workgroup” Of Twenty-FourProtein Targets Used To Test Platform Changes

David J. AcetiCenter for Eukaryotic Structural Genomics

University of Wisconsin-MadisonDepartment of Biochemistry

2009 NIGMS Workshop onEnabling Technologies for Structural Biology

March 5, 2009

The Control Workgroup is a selected set of proteins whose behavior is known at all stages of our platform.

The Control Workgroup has been used to:(1) Compare expression vectors(2) Compare methods of mRNA and plasmid preparation

for Wheat Germ Cell-Free expression(3) Compare E. coli and Cell-Free protein production

platforms (4) Compare growth medium formulations(5) Test new crystallization equipment(6) Test aspects of the purification process(7) Train staff

# ORF ID Protein Name Source Organism MW Assay

1 34382 cytoplasmic dynein light chain M. musculus 10990

2 6042 unknown protein At1g77540.1 A. thaliana 11736

3 14751 thioredoxin h1 A. thaliana 12673Spec assay following NADPH/insulin-DS redox reaction

4 33810 zinc finger protein H. sapiens 13169

5 81370 unknown heme-binding protein C. merolae 16474 Red-colored from heme binding

6 2361 unknown protein At1g01470.1 A. thaliana 16543

7 13193 unknown protein At3g03773.1 A. thaliana 17366

8 11624 thioredoxin-like protein A. thaliana 17947

9 91592 allene oxide cyclase variant1 A. thaliana 19522

10 91593 allene oxide cyclase variant2 A. thaliana 21232

11 35683 cysteine dioxygenase 1 M. musculus 23026 HLPC assay for cysteine sulfinic acid formation

12 605 phosphatase A. thaliana 24537 pNPP phosphatase assay

13 91571 Enhanced C3 green fluorescent protein A. victoria 26748 Green/fluorescent

14 91591 TEV protease Tobacco etch virus 26922 Fluoresence anisotropy-based protease assay

15 74368 Pre-mRNA processing factor 24 S. cerevisiae 27223 Gel mobility shift assay

16 80048sarcosine dimethylglycine

methyltransferase G. sulphuraria 33324Coupled spec. assay following deamination of adenine

17 37540 glyoxylate/hydroxypyruvate reductase H. sapiens 35668 NADPH-linked spectrophotometric assay

18 79368 aspartoacylase H. sapiens 35735Couple spec. assay following deamination of aspartic acid

19 70653 dimetal phosphatase D. rerio 36645 pNPP pPhosphatase assay

20 7312 putative steroid sulfotransferase A. thaliana 37140

21 8210 12-oxophytodienoate-10,11-reductase A. thaliana 42691 NADPH-dependent reduction of TNT

22 24674 agmatine iminohydrolase A. thaliana 43156 Assay of ammonia product by Bertelot reaction.

23 34351 unknown protein BC065058 M. musculus 50256

24 74329 Photinus (Firefly) luciferase P. pyralis 60844 Luciferase assay

The Control Workgroup

# ORF ID Protein Name MWWheat Germ Cell

Free History E. Coli Cell-Based History

1 34382 cytoplasmic dynein light chain 10990 NMR structure 1Y4O Small amount purified

2 6042 unknown protein At1g77540.1 11736 None NMR structure 2EVN . Xray structure 1XMT

3 14751 thioredoxin h1 12673 NMR structure 1XFL Small amount 15N purified

4 33810 zinc finger protein 13169 NMR structure 1ZR9 None

5 81370 unknown heme-binding protein 16474 Expressed soluble No crystals, HSQC-, good yield

6 2361 unknown protein At1g01470.1 16543 HSQC+ NMR structure 1XO8, high yields, crystal

7 13193 unknown protein At3g03773.1 17366 None Variable yields

8 11624 thioredoxin-like protein 17947 No expression NMR structure 1X0Y, high yields

9 91592 allene oxide cyclase variant1 19522 None Did not cleave

10 91593 allene oxide cyclase variant2 21232 Expressed soluble Xray structure 1Z8K

11 35683 cysteine dioxygenase 1 23026 None Xray structure 2ATF, moderate yield

12 605 phosphatase 24537 None Xray structure 1XRI, good yields

13 91571 Enhanced C3 green fluorescent protein 26748 None Xray structure 2QU1

14 91591 TEV protease 26922 None High yields

15 74368 Pre-mRNA processing factor 24 27223 None Xray structure 2GHP, high yields

16 80048 sarcosine dimethylglycine methyl-xase 33324 None Xray structure 2O57, very high yield

17 37540 glyoxylate/hydroxypyruvate reductase 35668 None Xray structure 2H1S, low yield

18 79368 aspartoacylase 35735 Expressed soluble Xray structure 2I3C, low yield

19 70653 dimetal phosphatase 36645 None Xray structure 2NXF, variable yields

20 7312 putative steroid sulfotransferase 37140 None Xray structure 1Q44

21 8210 12-oxophytodienoate-10,11-reductase 42691 None Xray structure 1Q45, high yield

22 24674 agmatine iminohydrolase 43156 Expressed soluble Xray structure 1VKP, very high yields

23 34351 unknown protein 50256 None Xray structure 2GNX, low yield

24 74329 Photinus (Firefly) luciferase 60844 None None

History of the Control Workgroup

Expression Vector Comparison

(1) Flexi® Cloning for both E. coli cell-based and wheat germ cell-free systems would allow great savings in time and money.

(2) Vectors with wildtype lacI are more compatible with autoinduction (Blommel et al., 2007, Biotech Prog, 23:585-598).

(3) A “self-cleaving” vector might cleave tags more efficiently and save time and money.

VectorCloning Method Antibiotic

Promotor/

RepressorTag

Cleavage Fusion Protein

Base VectorspVP16 (E. coli) Gateway Amp T5/lacIq TEV His- MBP -TEV-S- ORF

pEU-His (cell-free) Restriction N/A SP6 None His - XX- ORF

Experimental VectorspVP68K (E. coli) Flexi® Kan T5/lacI TEV His- MBP -3CP-TEV-S- ORF

pVP65K (E. coli) Flexi® Kan T5/lacI Self(TVMV) /TEV

MBP-TVMV-His-TEV-S-ORF

pEU-His-FV (cell-free) Flexi® N/A SP6 TEV His -TEV- S- ORF

0

4

8

12

16

20

24

28

32

Cloned Small-Scale

Large-Scale

FusionPurified

>100 mg

TargetPurified>1 mg

TargetPurified>10 mg

Crystals

pVP16

pVP68K

pVP65k

(lacI Flexi®)

(lacI Flexi® selfcleave)

(lacIq GW)

Suc

cess

Rat

e (#

Tar

gets

)

NA NA

Performance of 3 E. coli Expression Vectors in the Production of 24 SeMet-

labeled Control Workgroup Targets

0

4

8

12

16

Cloned Small-Scale

Large-Scale

FusionPurified>100 mg

TargetPurified>1 mg

TargetPurified>10 mg

HSQC+

pVP16

pVP68K

pVP65K

Performance of 3 E. coli Expression Vectors in the Production of 12

15N-labeled Control Workgroup Targets

NA NA

Suc

cess

Rat

e (#

Tar

gets

)

(lacI Flexi®)

(lacI Flexi®

selfcleave)

(lacIq GW)

15N Fusion SeMet Fusion 15N Target SeMet Target

pVP16 (lacIq) 133 184 9 19

pVP68K (lacI) 281 255 30 26

pVP65K (lacI self-cleaving)

38 80 2 4

Mean Yield (mg) of Fusion and Target Proteins from Three E. coli Vectors

# ORF ID Protein Name MW Historical pVP16 pVP68K Historical pVP16 pVP68K

1 34382 cytoplasmic dynein light chain 10990 X

2 6042 unknown protein At1g77540.1 11736

3 14751 thioredoxin h1 12673 X

4 33810 zinc finger protein 13169 X X X X

5 81370 unknown heme-binding protein 16474 X X X X In Progress X

6 2361 unknown protein At1g01470.1 16543

7 13193 unknown protein At3g03773.1 17366 X

8 11624 thioredoxin-like protein 17947 X X X

9 91592 allene oxide cyclase variant1 19522 X X X X

10 91593 allene oxide cyclase variant2 21232 X X X

11 35683 cysteine dioxygenase 1 23026 X X X

12 605 phosphatase 24537 X X X

13 91571 Enhanced C3 green fluorescent protein 26748

14 91591 TEV protease 26922 X X X

15 74368 Pre-mRNA processing factor 24 27223 X

16 80048 sarcosine dimethylglycine methyl-xase 33324

17 37540 glyoxylate/hydroxypyruvate reductase 35668

18 79368 aspartoacylase 35735

19 70653 dimetal phosphatase 36645 X

20 7312 putative steroid sulfotransferase 37140 X

21 8210 12-oxophytodienoate-10,11-reductase 42691

22 24674 agmatine iminohydrolase 43156

23 34351 unknown protein 50256 X

24 74329 Photinus (Firefly) luciferase 60844

E. coli Vectors: Historical vs. pVP16 vs. pVP68K15N HSQC + SeMet Crystallization +

0

4

8

12

16

20

24

28

Cloned Small-Scale

Large-Scale

Purified HSQC+

pEU-His-FV Uncleaved

pEU-His-FV Cleaved

Performance of a Wheat Germ Cell-FreeExpression Vector in the Production of

24 15N-labeled Control Workgroup Targets

Suc

cess

Rat

e (#

Tar

gets

)

ORF ID Protein Name MWHistoricalHSQC+

pEU-His-FVHSQC+

34382 cytoplasmic dynein light chain 10990

6042 unknown protein At1g77540.1 11736

14751 thioredoxin h1 12673

33810 zinc finger protein 13169

81370 unknown heme-binding protein 16474 X

2361 unknown protein At1g01470.1 16543 X

13193 unknown protein At3g03773.1 17366

11624 thioredoxin-like protein 17947 X X

91592 allene oxide cyclase variant1 19522

91593 allene oxide cyclase variant2 21232 X

35683 cysteine dioxygenase 1 23026

605 phosphatase 24537 X

91571 Enhanced C3 green fluorescent protein 26748

91591 TEV protease 26922 X

74368 Pre-mRNA processing factor 24 27223 X

80048 sarcosine dimethylglycine methyl-xase 33324 X

37540 glyoxylate/hydroxypyruvate reductase 35668 X

79368 aspartoacylase 35735 X X

70653 dimetal phosphatase 36645 X

7312 putative steroid sulfotransferase 37140 X

8210 12-oxophytodienoate-10,11-reductase 42691 X

24674 agmatine iminohydrolase 43156 X X

34351 unknown protein 50256 X

74329 Photinus (Firefly) luciferase 60844 X

Wheat Germ Cell-Free: Historical Vectors vs. Current Vector

Distinct Control Workgroup Targets Expressed Solubly from E. coli and Wheat Germ Cell-Free

E. coliWheat Germ Cell-Free

183 2

Distinct Arabidopsis Targets Expressed Solubly from E. coli and Wheat Germ Cell-Free

Tyler, R.C., et al. (2005) Comparison of cell-based and cell-free protocols for producingtarget proteins from the Arabidopsis thaliana genome for structural studies. Proteins 59:633-643

25 29 20

E. coli Wheat Germ Cell-Free

0

2

4

6

8

10

12

14

16

None Weak Moderate High

0

2

4

6

8

10

12

14

None Weak Moderate High

Expression Solubility

Old protocol

New protocol

None Weak Moderate High None Weak Moderate High

Use of Control Workgroup to test preparation of plasmidand mRNA for wheat germ cell-free production

Shin-ichi Makino

• mRNA coding for target protein is degraded by endogenous RNase activity carried over from plasmid template preparation, and this lowers the protein yield.

• mRNA has been concentrated by ethanol precipitation and is sometimes difficult to resolubilize, resulting in lower and less consistent protein yields.

Plasmids are now treated with proteases to inactivate RNase activity, and ethanol precipitation of mRNAhas been discontinued. Higher and more consistent levels of expression result as shown with the Control Workgroup.

# of

Tar

gets

• The Control Workgroup is a useful tool for comparing platform performance over a wide variety and a significant number of proteins.

• The lacI Flexi® vector pVP68K “does no harm” and produces greater quantities of protein. The Flexi Cloning system works for both E. coli and wheat germ platforms. This system saves time and money.

• The “self-cleaving” vector pVP65K is not viable; the concept needs further research.

• An improved method of preparing mRNA and plasmid for cell-free production was demonstrated.

* The Control Workgroup will be available shortly from the PSI Materials Repository as sets of twenty-four targets in 4 expression vectors.

Conclusions

Credit Goes To….Lai BergemanCraig Bingman Sethe BurgieMike CassidyClaudia CornilescuBrian FoxRonnie FrederickKasia GromekLeigh GrundhoeferAndrew LarkinBetsy LytleShin-ichi MakinoJohn MarkleyYuko MatsubaraKarl NicholsXiaokang Pan

Francis PetersonGeorge PhillipsMike PopelarsJohn PrimmGreg SabatSarata SahuKory SederDonna TroestlerFrank VojtikGary WesenbergRuss WrobelBrian VolkmanZsolt Zolnai

Many CESG Alumni …and many Students