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STRUCTURAL PROTEOMICS Structural biology on a genome-wide scale Cheryl Arrowsmith

STRUCTURAL PROTEOMICS Structural biology on a genome-wide scale Cheryl Arrowsmith Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto. Cloning and Test Expression. ligate. transform. clones. PCR x96. X 96. 24 x 3ml LB Kan, Amp 37C, Induce at OD600

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STRUCTURAL PROTEOMICS Structural biology on a genome-wide scale Cheryl Arrowsmith

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  1. STRUCTURAL PROTEOMICS Structural biology on a genome-wide scale Cheryl Arrowsmith Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto

  2. Cloning and Test Expression ligate transform clones PCR x96 X 96 24 x 3ml LB Kan, Amp 37C, Induce at OD600 Grow O/N 15C or 20C Kan, Amp X 96 300 ul 300 ul X 96 X 96 supernatant Spin, Dissolve pellet in SDS Spin, Freeze, Lyse with BugBusterTM Spin again SDS PAGE

  3. The NMR Pipeline 18 at a time in batch mode Steady state rate: 18 samples evaluated per week

  4. Feasibility Results for MT Proteins Christendat et al., 2000 > 20 kDa < 20kDa cloned expressed soluble purified Crystals or good/promising HSQC Well-diffracting crystals or good/promising HSQC 0 50 100 150 200 250 Number of Proteins

  5. Extension of our initial feasibility study

  6. Cloned E. coli Expressed Soluble yeast T. maritima Yee et al, PNAS, 2002 l phage Myxoma virus M. th.

  7. Yee et al, PNAS, 2002 15N-HSQC Results for Soluble Proteins PPT PROMISING POOR UNFOLDED GOOD M. th. yeast E. coli T. maritima Myxoma virus l phage

  8. Orthologues 68 Escherichia coli 68 Thermotoga maritima Topt 80 °C Topt 37 °C 1,860,725 bp 4,639,221 bp 1, 877 ORFs 4, 288 ORFs Expressed & soluble 62 48 Concentratable to > 2mg/ml 50 44 9 Proteins could not be purified from either species 15 35 9

  9. Total Crystals (30) T. maritima E. coli 11 3 13 Total Good/Promising NMR spectra (14) T. maritima E. coli 2 4 4

  10. NMR & Crystallography: complementary! 24 small proteins for which both crystal trials and NMR data collected Good/promising HSQC crystals 10 3 6 Of 32 proteins that gave poor HSQC’s 7 have crystallized

  11. E. Coli Expression Very good expression No expression Expression 150 M.th. Clones in Cell-Free Protein Expression System Total Protein % Soluble Protein Clone Shirouzu & Yokoyama, RIKEN

  12. Expressed Protein in Cell-Free System E. Coli Expression Soluble Not Soluble Total Protein Soluble Protein Clone Shirouzu & Yokoyama, RIKEN

  13. Data storage and Mining: Defined Vocabulary Property Vocabulary Expression level 0-5 (no expression – high expression) Solubility (test expression) 0-5 (insoluble – highly soluble) Concentratability 0-5 (or mg/ml) Crystal trials clear amorphous ppt granular ppt crystal Initial HSQC NMR good promising poor

  14. Ontario Cancer Institute Adelinda Yee (NMR) Brian Li Anthony Semesi Cheng-Song Liu Aled Edwards Limin Chen(Myxoma, l phage) Alexei Savchenko (T.m. E.coli) Tania Skarina Lena Evdokimova Marina Pavlova Nancy Ouellette Dinesh Christendat (M.th.) Diane Bona J. Gu Steven Beasley Micha Pennycooke Linda Wu

  15. Collaborators S. Yokoyama (RIKEN) Mikako Shirouzu Northeast Structural Genomics Consortium Midwest Structural Genomics Consortium

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