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March 7, 2002

March 7, 2002. NYSGRC NYSGXRC PI: Stephen K. Burley-CSO, SGX PI-of-Record: JB Bonanno, Rockefeller Mark R. Chance-AECOM S. Swaminathan-BNL Larry Shapiro-Columbia Medical School Andrej Sali-Rockefeller University Chris Lima-Weill Medical College Web Site: http://www.nysgrc.org/. NYC.

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March 7, 2002

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  1. March 7, 2002 NYSGRCNYSGXRCPI: Stephen K. Burley-CSO, SGX PI-of-Record: JB Bonanno, RockefellerMark R. Chance-AECOMS. Swaminathan-BNLLarry Shapiro-Columbia Medical SchoolAndrej Sali-Rockefeller UniversityChris Lima-Weill Medical CollegeWeb Site: http://www.nysgrc.org/

  2. NYC Target selection SGX Cloning & Expression SGX Fermentation SGX Protein Purification SGX/NYC Crystallization/Data Collection Structure Determination NYC NYC/SGX Division of Labor PDB

  3. Work Flow: NYCSGXNYCPublic Domain • Target Selection—Andrej Sali plus NYC Members • Uploading of Selected Targets from NYC ICE-DBSGX LIMS • Topoisomerase CloningE. coli • Small Scale Expression (N- and C-His, C-Smit3/His Tags) • Solubility Screening/Testing • Biophysical Characterization/Domain Mapping with MALDI-MS • Recloning of Various Constructs p.r.n. • Large Scale Expression/Purification (1 liter E. coli fermentation) • Quality Control/Quality Assurance (MS, DLS, CD, Fl, UV/Vis Abs) • Crystallization Screening (2 temps, incomplete factorial screens) • Diffraction Data Collection (APS, BNL) • Transfer of Reagents and SGX LIMS EntriesICE-DB in NYC • Structure Determination/Refinement/QA/QC/AnnotationPDB • All reagents and data will be made public via ICE-DB/NIH

  4. Exploiting Genomic Diversity in Target Selection Structural domains        Targets   Based on a 30% ID cutoff for good quality homology modeling      Species

  5. NYSGRC Mission Statement • Establish and exploit a technology platform for high-throughput protein structure determination with X-ray crystallography. • Targets are chosen from archaea, eubacteria, and eukaryotes with the goal of providing one experimental protein structure for each protein sequence family defined at the 30% identity levelPDB. • Preference is given to targets of high biological or medical relevance, provided that they also satisfy the 30% sequence identity criterion. • Perform high-throughput comparative protein structure (a.k.a. homology) modeling on all sequences within modeling distance (>30% identity) of each experimental structureMODBASE. • Provide accurate functional annotations and supply molecular biology/protein reagents to relevant experts.

  6. Topoisomerase Cloning of Expression Vectors • 5min ligation reactions at 20C • No requirement for restriction sites • Initial experience with His tags ($20/reaction) • N- and C-terminal +/- cleavage (polioviral protease) • TA Topo cloning used at SGX • Advantage: 90+% efficiency • Disadvantage: 50%:50% mixture • Directional Topo cloning used by NYSGRC • Advantage: 90+% inserts valid (x PCR errors) • Disadvantage: estimated 75% efficiency

  7. Chris Lima Vector: Double Tag Strategy • New fusion protein system with unique protease • Protein of Interest(POI)--Smit3--His double tag • Combined with directional Topo cloning • Developed, tested and patented by Chris Lima, WMC Co-PI for NYSGRC • Advantages: • Two stage purification: Ni and Ion Exchange • “Clean” removal of tags with protease that recognizes Smit3 and cuts at fusion with POI • Commercially available from Invitrogen soon • Technology transferred to SGX under lic. from WMC

  8. 96-Well Cloning and Protein Expression • N- and C- terminal His tags and Smit3/His Topoisomerase cloning • Qiagen 3000 & Qiagen 8000 and Beckman Biomek FX for PCR set-up, PCR fragment and plasmid purification with magnetic beads, cherry picking • Modified Genetix Q-Pik for colony picking • Genemachines Hi-Gro for E.coli expression at small scale (1-2ml) • Solubility screening in 96-well format • Clone confirmation by MALDI-MS following Ni ion purification with Zip-tips (1mg yield) • MALDI-MS/proteolysis (trypsin, AspN, GluC, chymotrypsin, subitlisin) for domain mapping (integration with bioinformatics)

  9. BL21(pLysIce) Improves Cell Lysis Yields Marker whole sup whole sup whole sup whole sup Marker POI LysIce f/t sonication LysS f/t RIL f/t Expression of Lambda lytic gene subset allows for freeze-thaw lysis Advantages: Faster, Cheaper, Gentler, 96-well Compatible

  10. Expression of 262 Bacterial Genes ProvesUtility of the Protein Families-Based Approach Species Af to Tm • Soluble proteins for 80% of gene sets • 40 Species • 1852 Gene Sets Gene number

  11. Protein Engineering by N/C Truncations of Kinase Oligo design _____ _______HHH DGKLYVSSES RFNTLAELVH HHSTVADGLI TTLHYPAPKR NKPTIYGVSP NYDKWEMERT + + + + + + ++++# + + + DITMKHKLGG GQYGEVYEGV WKKYSLTVAV KTLKEDTMEV EEFLKEAAVM KEIKHPNLVQ - LLGVCTREPP FYIITEFMTY GNLLDYLREC NRQEVSAVVL LYMATQISSA MEYLEKKNFI HRDLAARNCL VGENHLVKVA DFGLSRLMTG DTYTAHAGAK FPIKWTAPES LAYNKFSIKS DVWAFGVLLW EIATYGMSPY PGIDLSQVYE LLEKDYRMER PEGCPEKVYE LMRACWQWNP HHHHHH HHHHHHH_ SDRPSFAEIH QAFETMFQES SISDEVEKEL GKRGTRGGAG SMLQAPELPT KTRTCRRAAE ----++++ + +++ + + + + # + + Solubility screen Functional screen

  12. Phosphorylation Protein QA/QC-Mass Spectrometry • Analysis of all expressed proteins to verify termini and MW • More extensive characterization of eukaryotic proteins • Limited proteolysis & MS to define domain boundaries

  13. 6 MonoQ pH3 pH10 8 IEX 6 7 8 Phosphorylated Forms can be Separated by Ion-Exchange Chromatography – Human Kinase IEF gel MonoQ load M/s fraction 6

  14. ReSurface: Protein EngineeringCrystallizability(both gene shuffling and GFP methods tried) • Program that suggests mutations to alter protein solubility and crystallizability • First version finished, plan to explore SNPs, PPISP, crystal contacts • Accumulating data will be mined to improve method CFTR NBD1 domain resurface suggestions CONFIDENTIAL

  15. E. coli vs. Baculovirus SuggestsLow Threshold for Using Insect Cells Baculovirus: Mouse Kinase E. coli: Mouse Kinase E. Coli: C. Elegans Kinase • For this Kinase Set: • Less complex phosphorylation pattern from baculovirus • Single phosphorylation is same as in vivo • Bac-to-Bac System provides reasonable throughput

  16. Crystallization • Customized liquid handling robotics • Robbins 96- and single-channel pippettor • TECAN • Proprietary sitting drop plate • DLS on all samples prior to crystallization • Developing screens based on protein families and statistical analyses; using 4th generation screen; automated recipes • High-capacity storage system maintains 960,000 trials at each temperature • Automated imaging/scoring of crystals 96,000 trials analyzed/day/temperature

  17. Collaboration with Robodesign, Intl. • Completed December 2001 • RoboStore (10,000 plates) • RoboVision (programmable plate imaging and scoring)

  18. Integrated Storage/Imaging System Capacity 10,000 plates (approx. 106 trials) @ 2 temps Capability 96,000 trials Examined/day

  19. Fermentation and Purification • Fermentation at 1 liter scale • Overnight induction at 20C • Affinity purification on 5 ml nickel columns • BioCad and AKTA Explorer machines • Throughput is 60 – 75 pellets per week

  20. SGX-CAT at the APS (Sector 31) Collected first data sets in December 2001 Automation will be completed during Q2 2002 State-of-the-Art insertion device beamline Rapid data collection with small crystals High-redundancy SAD data collection Streamlined data reduction to |Fobs| T1 line to NYC (ICE-DB) will be installed during Q4 2002 Diffraction Data Collection at SGX

  21. SGX ID Beamlines(first 55 cases2.0 Å)NYSGRC BM/Wiggler (first 27 cases2.3 Å) Average Resolution = 2.0 Å, Range: 1.3 – 2.9 Å

  22. Indexing/Integration Sorting/Scaling Heavy atom location/Phasing Model building/Refinement Quality Control Analysis Automated Structure Determination Platform • ASDP: J-S Jiang, BNL • One day structure determinations: • <24 hours from data collection • to refinement of most (up to 95%) of the polypeptide chain • (limited by CPU speed and resolution limit of diffraction data)

  23. NYC and SGX Computational Platforms Target Selection, Homology Modeling Computational Chemistry • Fold Assignment • Domain Prediction • MODBASE/MOD* • Coding SNPs • DOCK5, AMBER • Virtual Screening pipeline Structure Annotation • Active site prediction • Classifying protein • active sites • Structure similarity • Distribution of • reagents to experts SGX LIMS & DataMining ReSurface Technology MALDI-MS/Domain Mapping Technology

  24. NYSGXRC Summary • Target Selection in Public Domain by NYC institutions • Industrial processes to be performed in industrial setting (SGX) • Transparent access to SGX-PSI progress reports via ICE-DB • Free distribution of reagents from NYC Institutions/NIH • Structure determination/annotation to occur in Public Domain • Intellectual property from structures owned by NYC Institutions • Full deposition and timely release of atomic coordinates, |Fs|, interim results to PDB, etc. by NYC Institutions (No SGX control!) • Benefits to PSI: leveraging SGX investment, increased throughput, roadmap leading towards the production phase • Benefits to SGX: continuous improvement of platform, pooling of SGX and PSI experiences for more rigorous statistical analyses, high PR value, high quality academic collaborations, potential benefit to the larger scientific community

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