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Structural Genomics of C. elegans

Structural Genomics of C. elegans. Ming Luo, Ph.D. University of Alabama at Birmingham March 7-8, 2002, NIH. C. elegans Genome. 19,092 genes vs. Human >31,000. Outcome = speed x success rate 100 crystals = 500 purified proteins x 20%

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Structural Genomics of C. elegans

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  1. Structural Genomics of C. elegans Ming Luo, Ph.D. University of Alabama at Birmingham March 7-8, 2002, NIH

  2. C. elegans Genome 19,092 genes vs. Human >31,000. Outcome = speed x success rate 100 crystals = 500 purified proteins x 20% 500 purified proteins = 2,000 expressed proteins x 25% 2,000 expressed proteins = 4,000 genes x 50% We have set-up a system to process 350 genes per 15 days. ~4,500 genes per year

  3. Expression System GATEWAY Cloning (Invitrogen) 1. cDNAs in ENTRY vectors. 2. Three vectors pDEST17.1 - N-His-tag pET15b - N-His-tag/Thrombin site pET11a - N- 7aa-tag Others 3. Biomek FX, Qiagen 3000 Biomek 2000 (x 2)

  4. Workflow • cDNA clone by Gateway (Vidal at MGH) • LR reaction in 96-well • Transform into DH5a in 96-well • Pick single colonies, 1ml culture and miniprep plasmids • Transform into BL21(DE3) in 96-well • Pick single colonies, 1 ml culture and induce 3 hours (37°C) or 20 hours (18°C)

  5. continue • Lyse cells with or without 8M urea • Filter debris in 96-well, centrifugation • Ni-beads to purify the protein • ELISA for detection of His-tag • 1L culture for soluble clones, then 6L • Others go through refolding • Purification by Ni-column and S-75

  6. Refolded Protein 1. MW in non reducing SDS. 2. Single peak from ion exchange column.

  7. Current Progress http://sgce.cbse.uab.edu *Note: % is calculated per group of proteins under study.

  8. Expression Profiling

  9. Jim Finley, Ph.D. Songlin Li, Ph.D. Danlin Luo, Ph.D. Mike Carson, Ph.D. Jun Tsao, Ph.D. Lisa Nagy, Ph.D. Chi-Hao Luan, Ph.D. Henry Symersky, ph.D. Bingdong Sha, Ph.D. Larry DeLucas, Ph.D. ShiHong Qiu David H. Johnson Jun Zhao GuangDa Lin Willie Thomas Alexis Stanton Jason Davis Wenying Huang Rita Gray Debra Brady Acknowledgement- UAB Group

  10. 1 2 3 F11 29kD+tags F11 29kD+tags 34.8kD 34.8kD Trypsinized Fragments F53F4.3

  11. F53F4.3 Crystal Space Group: P6122 a = 64.26 Å b = 64.26 Å c = 102.39 Å 1 Mol/A.U. Resolution: 1.85 Å Rsym: 0.042 (0.712) I/sigma: 21.3 (4.6),

  12. F54F4.3 Structure Structure determined by 2.5 Å sulfur SAS phasing Refined to 1.77 Å resolution

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