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Eukaryotic Gene Families Coverage of Structure Space Prioritization by Functional Genomics

Eukaryotic Gene Families Coverage of Structure Space Prioritization by Functional Genomics Complementarity of NMR and Xray Crystallography. Protein Production Technologies for NESG Consortium. E. coli Production Vectors pET based with HexaHis Tags - multiplex vector set

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Eukaryotic Gene Families Coverage of Structure Space Prioritization by Functional Genomics

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  1. Eukaryotic Gene Families Coverage of Structure Space Prioritization by Functional Genomics Complementarity of NMR and Xray Crystallography

  2. Protein Production Technologiesfor NESG Consortium E. coli Production Vectors pET based with HexaHis Tags - multiplex vector set - manual cloning with robotic assistance for PCR - 96-well format, Qiagen Robot Mgk, Rosetta, and CodonPlus Codon-supplemented Strains GateWay Ligase-free Cloning Maltose-Binding Protein Fusions CAT Fusion Proteins Cold Shock Promoters Templates Genomic templates cDNA clones RT PCR Fermentation Technologies Heat Shock (42 deg C) Low Temperature Fermentation (17 deg C) Cell Free Protein Production

  3. Dr. Tom Acton, Ph.D. Prof. Inouye Masayori, Ph.D. Prof. Gaetano Montelione, Ph.D. Molecular Biology Ritu Shastry, M.S. Margaret Wu, B.S. Bonnie Cooper Nodia Khan Fermentation and Protein Chemistry Yiwen Chiang, M.S. Teresa Climent, M.S. Rong Xiao, M.S. Kate Drahos Laura Lee Rebecca Liu Lydia Shih New Technologies LiChung Ma, Ph.D. Helen Chow

  4. Rost Clusters “Clusters of Homologous Proteins” • pairwise seq id greater than about 30% • no represenatives in PDB. • small (< 340 AA) full length proteins Each of the Rost Clusters has at least one homologue in Target Eukaryotic Genomes: human, worm, fly, yeast, (arabidopsis) Samples prepared from Reagent Genomes: human, worm, fly, yeast, E coli, M. thermoauto, T. maritima, B. subtilis, A. aeolicus, A. thalia, etc.

  5. A. aeolicus A. thaliana B. subtilis C. elegans D. melanogaster E. coli H. influenzae H. pylori S. cerevisiae S. aureus T. thermophilus NESG Target List Phylogenetic Distribution 1% 2% 1% 24% 38% worm yeast fly 1% 21% 4% 8% 1879 Rost Cluster Targets + ~ 20 Technology Development Targets - MTH

  6. There are over 2400 Protein Targets in SPINE Mark Gerstein, et al.

  7. Zeba Wunderlich, Rutgers College ‘03

  8. Multiplexed Construct Generation Classical Restriction Enzyme / Ligation cloning

  9. Gateway Recombinational Cloning Recombinational Cloning - efficient, fast, less steps - extensive Gateway libraries - avoid ligation - high efficiency or avoid colony PCR Using the existing destination vectors and libraries of cloned ORFs 15 extraneous N-term amino acids + HexaHis - 8 from AttB site - 7 from Invitrogen constuction Can affect with solubility and complicate structure determination Engineer in protease cleavage site - Adds PCR steps - Cannot go straight from available Gateway ORF Libraries - Efficiency of cleavage - Additional steps in purification - Added complexity 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

  10. MJ Minimal Media for Isotope Enrichment Jansson, M.; Li, Y.-C.; Jendeberg, L.; Anderson, S.; Montelione, G.T.; Nilsson, B. J. Biomol. NMR 1996, 7: 131 - 141. High level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. Works well when growing bacteria in 95% 2H2O Works well for SeMet labeling when supplemented with amino acids to suppress metabolic shuffling

  11. Screening for Protein “Foldedness”1H - 15N HSQC Spectra 15N 1H ppm 1H ppm T. thermophilus BRCT domain D. melanogaster P1CT domain

  12. Protein Production for X-Ray Crystallography and NMR Studies Analytical Gel Filtration with Static / Dynamic Light Scattering Protein Purification with Ni-NTA Affinity Column Protein Expression using 15N-MJ Media Pure 6xHis-Tagged Protein (> 20 mg) Ion Exchange (optional) Preparative Gel Filtration under Monodisperse Conditions HSQC (~ 3 mg) Robotic Crystallization Trials (~ 8 mg) Manual Crystallization Trials (~ 8 mg) CD Spectroscopy (~ 1 mg) SDS-PAGE and Mass Spec 10 - 20 mg 13C,15N or 2H,13C,15N-enriched Protein for NMR Studies Crystals 20 mg SeMet-enriched Protein for Crystallization Optimization and X-Ray Crystallography

  13. Production Results: Rost Targets at Rutgers CABM T. Acton, Y. Chiang, T. Climent, K. Gunsalus, D. Palacios, M. Wu, R. Xiao, et al

  14. Robotic Platform for Classical Restriction Enzyme - Ligase Cloning Qiagen BioRobot 8000

  15. Restriction Enzyme / Ligase Based Cloning Template Colony PCR cDNA synthesis (RT-PCR) Plate / Colony Pick DGC1.0 (384 well plates) Genomic DNA Transformation Gel-Loading PCR-set up Ligation PCR cleanup Mini-prep Glycerol stock Archiving Expression Screening Purification of Cut PCR Product RE digestion

  16. PCR - Set up usingBioRobot 8000 Drosophila Targets Product obtained with Correct size A1 - B12 Bacillus subtillis (30) 95% Aquifex aeolicus (20) 91% Archea (60) 93% Drosophila melanogaster (60) 80% C1 - D12 Drosophila Gene Collection (DGC 1.0) Rubin et al., Science 2000

  17. Concentrating Inserts Lyophilization Resuspend at high concentration in H2O Concentration of inserts (cleaved, purified PCR products of the ORFS) is critical for ligation process. - Low conc. from Qiaquick purification - Possible buffer effects (high pH for elution) Manual cloning - concentration by ethanol precipitation greatly improved ligation efficiency - concentration - further purification Tried volatile buffer to elute from Qiaquick - poor results. Desalt by Size Exclusion using 96-well “Big Dye” removal plates Low volume, salt free, high concentration DNA inserts

  18. Challenges to The Protein Production Process Li-Chung Ma, Ph.D. Center for Advanced Biotechnology and Medicine Rutgers, The State University of New Jersey

  19. Challenges to The Protein Production Process • cDNA clones or templates for Eukaryotic systems • Develop a htp screening of protein solubility • Optimize fermentation conditions and improve fermentation facilities • Solubilize proteins expressed at very low solubility • Evaluate the effects of purification tags on crystallization • Develop general parallel approaches for protein purification

  20. cDNA clones or templates for Eukaryotic systems -cDNA-Template for PCR Bottleneck for cloning Eukaryotic genes 1. cDNA Libraries -PolyT Priming 3' UTR not full length ORFs2. Availability -not always easily accessible3. Not fully sequenced -not consistently full length4. Representative - not all tissue/development etc. 5. Transfer- 384 -Well plates need special consideration for reformatting to 96- well plates - Direct cloning from total mRNA using RT-PCR

  21. cDNA Cloning Using RT-PCR A. Extract RNA (total) B. Reverse Transcription from mRNA 3' gene specific primer C. Add 5' Primer, etc. D. Normal PCR

  22. Results from RT-PCR Using Total RNA of Arabidopsis thalianaAs Templates Arabidopsis thaliana 1. cDNA Synthesis 50% isolated / cloned 2. Leaf / Stem Total RNA primers AR13 AR14 AR15 AR16 AR17 1 Kb Disadvantages 1. Gene must be expressed under conditions RNA was harvested a. Tissue specific b. Developmental-stage specific c. Stimulus dependent 2. Even if expressed, mRNA transcript level may be very low 3. More difficult procedure

  23. Develop a htp screening of protein solubility

  24. Multiplex Expression - NS1 C-term Domain mg / liter

  25. Cell-free Synthesis of GFP Using 5 Different Templates 5’C15Ambion / 3’C15T7terminator* 5’Ambion / 3’C15T7terminator* 5’Roche / 3’C15T7terminator* 5’A6 / 3’C15T7terminator* Roche GFP control vector 1 2 3 4 5 GFP (C)15GGATCC GFP GGATCC GAAAT AAAAAA pIVEXGFP * No. 1-4 are linear PCR templates, containing GFP gene, with different 5’ ends † S30 extract was prepared from BL21 Star w/ Rosetta

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