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A “Control Workgroup” Of Twenty-Four Protein Targets Used To Test Platform Changes David J. Aceti Center for Eukaryotic Structural Genomics University.

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Presentation on theme: "A “Control Workgroup” Of Twenty-Four Protein Targets Used To Test Platform Changes David J. Aceti Center for Eukaryotic Structural Genomics University."— Presentation transcript:

1 A “Control Workgroup” Of Twenty-Four Protein Targets Used To Test Platform Changes David J. Aceti Center for Eukaryotic Structural Genomics University of Wisconsin-Madison Department of Biochemistry 2009 NIGMS Workshop on Enabling Technologies for Structural Biology March 5, 2009

2 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

3 #ORF IDProtein NameSource OrganismMWAssay cytoplasmic dynein light chainM. musculus unknown protein At1g A. thaliana thioredoxin h1A. thaliana12673 Spec assay following NADPH/insulin-DS redox reaction zinc finger proteinH. sapiens unknown heme-binding proteinC. merolae16474Red-colored from heme binding 62361unknown protein At1g A. thaliana unknown protein At3g A. thaliana thioredoxin-like proteinA. thaliana allene oxide cyclase variant1A. thaliana allene oxide cyclase variant2A. thaliana cysteine dioxygenase 1M. musculus23026HLPC assay for cysteine sulfinic acid formation 12605phosphataseA. thaliana24537pNPP phosphatase assay Enhanced C3 green fluorescent proteinA. victoria26748Green/fluorescent TEV proteaseTobacco etch virus26922Fluoresence anisotropy-based protease assay Pre-mRNA processing factor 24S. cerevisiae27223Gel mobility shift assay sarcosine dimethylglycine methyltransferaseG. sulphuraria33324 Coupled spec. assay following deamination of adenine glyoxylate/hydroxypyruvate reductaseH. sapiens35668NADPH-linked spectrophotometric assay aspartoacylaseH. sapiens35735 Couple spec. assay following deamination of aspartic acid dimetal phosphataseD. rerio36645pNPP pPhosphatase assay putative steroid sulfotransferaseA. thaliana oxophytodienoate-10,11-reductaseA. thaliana42691NADPH-dependent reduction of TNT agmatine iminohydrolaseA. thaliana43156Assay of ammonia product by Bertelot reaction unknown protein BC065058M. musculus Photinus (Firefly) luciferaseP. pyralis60844Luciferase assay The Control Workgroup

4 #ORF IDProtein NameMW Wheat Germ Cell Free HistoryE. Coli Cell-Based History cytoplasmic dynein light chain10990NMR structure 1Y4OSmall amount purified 26042unknown protein At1g NoneNMR structure 2EVN. Xray structure 1XMT thioredoxin h112673NMR structure 1XFLSmall amount 15N purified zinc finger protein13169NMR structure 1ZR9None unknown heme-binding protein16474Expressed solubleNo crystals, HSQC-, good yield 62361unknown protein At1g HSQC+NMR structure 1XO8, high yields, crystal unknown protein At3g NoneVariable yields thioredoxin-like protein17947No expressionNMR structure 1X0Y, high yields allene oxide cyclase variant119522NoneDid not cleave allene oxide cyclase variant221232Expressed solubleXray structure 1Z8K cysteine dioxygenase NoneXray structure 2ATF, moderate yield 12605phosphatase24537NoneXray structure 1XRI, good yields Enhanced C3 green fluorescent protein26748NoneXray structure 2QU TEV protease26922NoneHigh yields Pre-mRNA processing factor NoneXray structure 2GHP, high yields sarcosine dimethylglycine methyl-xase33324NoneXray structure 2O57, very high yield glyoxylate/hydroxypyruvate reductase35668NoneXray structure 2H1S, low yield aspartoacylase35735Expressed solubleXray structure 2I3C, low yield dimetal phosphatase36645NoneXray structure 2NXF, variable yields putative steroid sulfotransferase37140NoneXray structure 1Q oxophytodienoate-10,11-reductase42691NoneXray structure 1Q45, high yield agmatine iminohydrolase43156Expressed solubleXray structure 1VKP, very high yields unknown protein50256NoneXray structure 2GNX, low yield Photinus (Firefly) luciferase60844None History of the Control Workgroup

5 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: ). (3) A “self-cleaving” vector might cleave tags more efficiently and save time and money. Vector Cloning MethodAntibiotic Promotor/ Repressor Tag CleavageFusion Protein Base Vectors pVP16 (E. coli) Gateway AmpT5/lacI q TEV His- MBP -TEV-S- ORF pEU-His (cell-free) Restriction N/ASP6None His - XX- ORF Experimental Vectors pVP68K (E. coli) Flexi ® KanT5/lacITEV His- MBP -3CP-TEV-S- ORF pVP65K (E. coli) Flexi ® KanT5/lacISelf (TVMV) /TEV MBP-TVMV-His-TEV-S-ORF pEU-His-FV (cell-free)Flexi ® N/ASP6TEV His -TEV- S- ORF

6 (lacI Flexi ® ) (lacI Flexi ® selfcleave) (lacI q GW) Success Rate (# Targets) NA Performance of 3 E. coli Expression Vectors in the Production of 24 SeMet- labeled Control Workgroup Targets

7 Performance of 3 E. coli Expression Vectors in the Production of N-labeled Control Workgroup Targets NA Success Rate (# Targets) (lacI Flexi ® ) (lacI Flexi ® selfcleave) (lacI q GW)

8 15 N Fusion SeMet Fusion 15 N TargetSeMet Target pVP16 (lacI q ) pVP68K (lacI) pVP65K (lacI self-cleaving) Mean Yield (mg) of Fusion and Target Proteins from Three E. coli Vectors

9 # ORF IDProtein NameMWHistoricalpVP16pVP68KHistoricalpVP16pVP68K cytoplasmic dynein light chain10990X 26042unknown protein At1g thioredoxin h112673X zinc finger protein13169XXXX unknown heme-binding protein16474XXXXIn ProgressX 62361unknown protein At1g unknown protein At3g X thioredoxin-like protein17947XXX allene oxide cyclase variant119522XXXX allene oxide cyclase variant221232XXX cysteine dioxygenase XXX 12605phosphatase24537XXX Enhanced C3 green fluorescent protein TEV protease26922XXX Pre-mRNA processing factor X sarcosine dimethylglycine methyl-xase glyoxylate/hydroxypyruvate reductase aspartoacylase dimetal phosphatase36645X putative steroid sulfotransferase37140X oxophytodienoate-10,11-reductase agmatine iminohydrolase unknown protein50256X Photinus (Firefly) luciferase60844 E. coli Vectors: Historical vs. pVP16 vs. pVP68K 15 N HSQC +SeMet Crystallization +

10 Performance of a Wheat Germ Cell-Free Expression Vector in the Production of N-labeled Control Workgroup Targets Success Rate (# Targets)

11 ORF IDProtein NameMW Historical HSQC+ pEU-His-FV HSQC cytoplasmic dynein light chain unknown protein At1g thioredoxin h zinc finger protein unknown heme-binding protein16474X 2361unknown protein At1g X 13193unknown protein At3g thioredoxin-like protein17947XX 91592allene oxide cyclase variant allene oxide cyclase variant221232X 35683cysteine dioxygenase phosphatase24537X 91571Enhanced C3 green fluorescent protein TEV protease26922X 74368Pre-mRNA processing factor X 80048sarcosine dimethylglycine methyl-xase33324X 37540glyoxylate/hydroxypyruvate reductase35668X 79368aspartoacylase35735XX 70653dimetal phosphatase36645X 7312putative steroid sulfotransferase37140X oxophytodienoate-10,11-reductase42691X 24674agmatine iminohydrolase43156XX 34351unknown protein50256X 74329Photinus (Firefly) luciferase60844X Wheat Germ Cell-Free: Historical Vectors vs. Current Vector

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

13 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 producing target proteins from the Arabidopsis thaliana genome for structural studies. Proteins 59: E. coli Wheat Germ Cell-Free

14 Expression Solubility Old protocol New protocol None Weak Moderate High Use of Control Workgroup to test preparation of plasmid and 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 mRNA has been discontinued. Higher and more consistent levels of expression result as shown with the Control Workgroup. # of Targets

15 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

16 Credit Goes To…. Lai Bergeman Craig Bingman Sethe Burgie Mike Cassidy Claudia Cornilescu Brian Fox Ronnie Frederick Kasia Gromek Leigh Grundhoefer Andrew Larkin Betsy Lytle Shin-ichi Makino John Markley Yuko Matsubara Karl Nichols Xiaokang Pan Francis Peterson George Phillips Mike Popelars John Primm Greg Sabat Sarata Sahu Kory Seder Donna Troestler Frank Vojtik Gary Wesenberg Russ Wrobel Brian Volkman Zsolt Zolnai Many CESG Alumni …and many Students

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