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  1. Production of proteins from Pyrococcus furiosus, C. elegans and human Mike Adams, Ming Luo and Harry Dailey of Southeast Collaboratory for Structural Genomics (SECSG) B.-C. Wang, Mike Adams, Harry Dailey, Jim Prestegard and John Rose (University of Georgia) and Ming Luo and Larry DeLucas (University of Alabama, Birmingham)

  2. Pyrococcus furiosus - Model Procaryotic Genome (small genome size, evolutionary position) Hyperthermophilic archaeon (archaebacterium) Genome of 1.9 Mb, encoding 2,236 ORFs 45% “known” (show similarity to characterized proteins) 45% unknown/hypothetical, 10% unique to Pfu Targets? All 2,236 ORFs - include representatives of most/all protein types (?) Generate functional proteins for structural analyses Correlate structural data with functional analyses (microarray, two hybrid, metabolic, biochemical, etc.) of native organism

  3. Not all ORFs are created equal! (expression-wise) Classification of ~2,200 ORFs of P. furiosus * ** * 1/3rd of all proteins contain metals? ** 23% of ORFs have transmemb. helices *** Approx. 600 mORFs ***

  4. Model System for HTP-Expression: Pyrococcus furiosus P. furiosus GENOME Beckman BioMek FX Bar-coded, 2,800 clone library [Custom pET, C-His, pRIL] Beckman BioMek FX 5 L Fermentor (2) 15 ( N 1 L Fermentor) Purification (x2) Purification (x10) (His/Q/S/SDS) (Hisx2/SDS) ≤50 mg/1 ml ≤5 mg/100 µl 5 proteins/week 3 0 proteins/week NMR/crystallography NMR/crystallography Structures Screening 2,200 sORFS + 600 mORFs HTP Cloning Module HTP-Expression Module Optimize Expression Conditions (Small Scale) by a Reiterative Robotic Approach Standard Expression Protocol (I) HTP Small Scale Expression (SSE, 1 ml) (96-well, 12 growth conditions, anti-His) SolubleInclusion Body Lethal No Express. Membrane no Alternative Expression Protocols II, III, IV and V Mass. Spec. yes Preparative 1 L Flasks (x10) Large Scale Expression (LSE, 1 liter) no Quality Control yes Analytical yes

  5. Sol (soluble fraction) KCl (salt wash) IB (urea wash) Mem (SDS wash) Small Scale Expression ELISA Analysis Expressed? How much? Which fraction? 1 ml cultures (96 well) Fractionation ELISA Expression Vector Custom pET with restriction sites(BamHI, NotI) and N-terminal (His)6-tag (E. coli, RIL) M--A--H--H--H--H--H--H--G--S--PROTEIN

  6. Sol: Soluble fraction IB: 8M Urea wash NE: No expression KCl: 3 M KCl wash Mem: SDS wash Blank Results of ELISA: Condition 1 (25˚C/0.4 mM IPTG) Sol KCl IB Mem NE

  7. Sol: Soluble fraction IB: 8M Urea wash NE: No expression KCl: 3 M KCl wash Mem: SDS wash Blank Results of ELISA - 4 conditions Condition 1 (25˚C/0.4mM) Condition 2 (25˚C/1.0mM) Condition 3 (37˚C/0.4mM) Condition 4 (37˚C/1.0mM) Sol KCl IB Mem NE

  8. Soluble (Condition 1) Summary of ELISA results for Project 11 Blank Condition 1

  9. Soluble (Condition 1) Summary of ELISA results for Project 11 Soluble (Condition 2) Blank Condition 1 + 2

  10. Soluble (Condition 1) Summary of ELISA results for Project 11 Soluble (Condition 3) Soluble (Condition 2) Blank Condition 1 + 2 + 3

  11. Soluble (Condition 1) Summary of ELISA results for Project 11 Soluble (Condition 3) Soluble (Condition 2) Soluble (Condition 4) Blank Condition 1 + 2 + 3 + 4

  12. Soluble Not Expressed Inclusion Body Cloning failure/Control IB Sol. IB Mem. NE 79/89 0/89 9/89 1/89 Summary of ELISA results for Project 11 - Small Scale Expn

  13. Soluble Not Expressed Inclusion Body Cloning failure/Control IB Sol. IB Mem. NE 79/89 0/89 9/89 1/89 Summary of ELISA results for Project 11 - Small Scale Expn

  14. Project 11 - Large Scale Expression (1 liter) (represented in 96 well format) 60 of 89 (67%) clones produce protein on large scale using 4 conditions

  15. Project 11 - Large Scale Expression (1 liter) 60 of 89 (67%) original clones produce protein on large scale using 4 different conditions

  16. Project 11 - ORF Classification (InterPro) 59/94 (63%) of ORFs ‘unknown’ InterPro:Integrated resource of protein families, domains and motifs. Includes SWISS-PROT, PROSITE, PRINTS, ProDom, Pfam, SMART and TIGRFAMs (www.ebi.ac.uk/interpro/). Putative metal binding motifs are mainly Cys-X-X-Cys.

  17. Quality Control/Characterization Purity - SDS-PAGE analysis (≥ 95%) Yield - for crystal and NMR screen (0.5 ml of ≥ 0.3 mM) Metal Content - ICP-MS (Fe, Zn, Cu, Cr, Co, Ni, etc.) Identity - Mass spectrometry

  18. Metal Analysis ICP-MS (ppb): Fe, Zn, Cu, Cr, Co, Ni, Cd, Mg, W, Mn, Mo ‘Positive’ considered > 0.1 atoms/monomer

  19. Metal leaching from affinity columns: Ni vs. Co Co contamination common, Ni contamination rare(?)

  20. Mass Spectrometry (ESI)(10 of 61 proteins - significant differences) 60000 50000 40000 Actual mass (Da) 30000 20000 10000 0 0 10000 20000 30000 40000 50000 60000 Predicted mass (Da)

  21. Mass Spectrometry (ESI)(15 clones sequenced, 13 were correct) 60000 50000 + 893 Da (recombined) 40000 Actual mass (Da) 30000 - 9,000 Da (wrong clone) 20000 10000 - 7,147 Da (degraded) 0 0 10000 20000 30000 40000 50000 60000 Predicted mass (Da)

  22. Summary: Production of P. furiosus proteins Total ORFs:2,836 (sORFs + mORFs) PCR products:1,823 (#1-20) ORFs cloned: 1,465 ORFs expressed (large, 1 liter):242 (#1,3,5,6,11) Proteins purified:200 (83%) Proteins soluble: 169 (85%) Proteins ppt’d: 31 (15%) (Proteins in progress, QC: 124) Proteins for crystal screen :45 Proteins crystallized: 24 (53%)

  23. Summary: Production of P. furiosus proteins Total ORFs:2,836 (sORFs + mORFs) PCR products:1,823 (#1-20) ORFs cloned: 1,465 ORFs expressed (large, 1 liter):242 (#1,3,5,6,11) Proteins purified:200 (83%) Proteins soluble: 169 (85%) Proteins ppt’d: 31 (15%) (Proteins in progress, QC: 124) Proteins for crystal screen:45 Proteins crystallized: 24 (53%) Acknowledgements: Frank Jenney*, Phil Brereton*, Claudia Shah, Frank Sugar, Michi Izumi, Farris Poole (IT), Brian Gerwe (UG), John Mackert (UG)

  24. C. elegans Ming Luo* Jim Finley* Songlin Li Danlin Luo Mike Carson Jun Tsao Lisa Nagy Chi-Hao Luan Henry Symersky Bingdong Sha Larry DeLucas Acknowledgements P. furiosus Mike Adams* Frank Jenney* Phil Brereton* Claudia Shah Frank Sugar Michi Izumi Farris Poole Human Harry Dailey* Tamara Dailey Mike Mayer *In the audience