Structural Genomics of C. elegans

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Structural Genomics of C. elegans

• Ming Luo, Ph.D.
• University of Alabama at Birmingham
• March 7-8, 2002, NIH

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C. elegans Genome
19,092 genes vs. Human gt31,000. 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
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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)
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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 (37C) or 20 hours (18C)

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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

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Refolded Protein
1. MW in non reducing SDS. 2. Single peak from
ion exchange column.
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Current Progress
http//sgce.cbse.uab.edu
Note is calculated per group of
proteins under study.
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Expression Profiling
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Acknowledgement- UAB Group
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F53F4.3
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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),
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F54F4.3 Structure
Structure determined by 2.5 Å sulfur SAS
phasing Refined to 1.77 Å resolution
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