1 / 28

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.

totie
Télécharger la présentation

March 7, 2002

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related