Robotic Platform for Classical Restriction Enzyme/Ligase Cloning

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Eukaryotic Gene Families Coverage of Structure
Space Prioritization by Functional
Genomics Complementarity of NMR and Xray
Crystallography
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Protein Production Technologiesfor NESG
Consortium

• E. coli Production Vectors
• pET based with HexaHis Tags
• - multiplex vector set
• - manual cloning with robotic assistance for
  PCR
• - 96-well format, Qiagen Robot
• Mgk, Rosetta, and CodonPlus Codon-supplemented
  Strains
• GateWay Ligase-free Cloning
• Maltose-Binding Protein Fusions
• CAT Fusion Proteins
• Cold Shock Promoters
• Templates
• Genomic templates
• cDNA clones
• RT PCR
• Fermentation Technologies
• Heat Shock (42 deg C)
• Low Temperature Fermentation (17 deg C)
• Cell Free Protein Production

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Dr. Tom Acton, Ph.D. Prof. Inouye Masayori,
Ph.D. Prof. Gaetano Montelione, Ph.D. Molecular
Biology Ritu Shastry, M.S. Margaret Wu,
B.S. Bonnie Cooper Nodia Khan Fermentation and
Protein Chemistry Yiwen Chiang, M.S. Teresa
Climent, M.S. Rong Xiao, M.S. Kate Drahos Laura
Lee Rebecca Liu Lydia Shih New
Technologies LiChung Ma, Ph.D. Helen Chow
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Rost Clusters

• Clusters of Homologous Proteins
• pairwise seq id greater than about 30
• no represenatives in PDB.
• small (lt 340 AA) full length proteins
• Each of the Rost Clusters has at least one
  homologue in Target Eukaryotic Genomes human,
  worm, fly, yeast, (arabidopsis)
• Samples prepared from Reagent Genomes human,
  worm, fly, yeast, E coli, M. thermoauto, T.
  maritima, B. subtilis, A. aeolicus, A. thalia,
  etc.

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NESG Target List Phylogenetic Distribution
1
2
1
24
38
worm
yeast
fly
1
21
4
8
1879 Rost Cluster Targets 20 Technology
Development Targets - MTH
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There are over 2400 Protein Targets in SPINE
Mark Gerstein, et al.
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Zeba Wunderlich, Rutgers College 03
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Multiplexed Construct Generation
Classical Restriction Enzyme / Ligation cloning
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Gateway Recombinational Cloning

• Recombinational Cloning
• - efficient, fast, less steps
• - extensive Gateway libraries
• - avoid ligation
• - high efficiency or avoid colony PCR
• Using the existing destination vectors and
• libraries of cloned ORFs
• 15 extraneous N-term amino acids HexaHis
• - 8 from AttB site
• - 7 from Invitrogen constuction
• Can affect with solubility and
• complicate structure determination
• Engineer in protease cleavage site
• - Adds PCR steps
• - Cannot go straight from
• available Gateway ORF Libraries

1 2 3 4 5 6 7 8
9 10 11 12 13 14 15
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MJ Minimal Media for Isotope Enrichment
Jansson, M. Li, Y.-C. Jendeberg, L. Anderson,
S. Montelione, G.T. Nilsson, B. J. Biomol.
NMR 1996, 7 131 - 141. High level production
of uniformly 15N- and 13C-enriched fusion
proteins in Escherichia coli. Works well when
growing bacteria in 95 2H2O Works well for
SeMet labeling when supplemented with amino
acids to suppress metabolic shuffling
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Screening for Protein Foldedness 1H - 15N HSQC
Spectra
15N
1H ppm
1H ppm
T. thermophilus BRCT domain D.
melanogaster P1CT domain
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Protein Production for X-Ray Crystallography and
NMR Studies
Analytical Gel Filtration with Static / Dynamic
Light Scattering
Protein Purification with Ni-NTA Affinity Column
Protein Expression using 15N-MJ Media
Ion Exchange (optional)
Pure 6xHis-Tagged Protein (gt 20 mg)
Preparative Gel Filtration under Monodisperse
Conditions
HSQC ( 3 mg)
Robotic Crystallization Trials ( 8 mg)
Manual Crystallization Trials ( 8 mg)
CD Spectroscopy ( 1 mg)
SDS-PAGE and Mass Spec
10 - 20 mg 13C,15N or 2H,13C,15N-enriched
Protein for NMR Studies
Crystals
20 mg SeMet-enriched Protein for Crystallization
Optimization and X-Ray Crystallography
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Production Results Rost Targets at Rutgers CABM
Worm Eubacteria Total
Cloned 83 187 270 (10 / wk)
Expressed and Soluble 35 (42) 88 (47) 123 (45)
Scaled Up and Purified 30 71 101
Crystals 7 (23) 11 (15) 18 (18)
Good HSQC 9 (30) 9 (13) 18 (18)
T. Acton, Y. Chiang, T. Climent, K. Gunsalus,
D. Palacios, M. Wu, R. Xiao, et al
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Robotic Platform for Classical Restriction
Enzyme - Ligase Cloning
Qiagen BioRobot 8000
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Restriction Enzyme / Ligase Based Cloning
Template
Colony PCR
cDNA synthesis (RT-PCR)
Plate / Colony Pick
DGC1.0 (384 well plates)
Genomic DNA
Transformation
Gel-Loading
PCR-set up
Ligation
PCR cleanup
Mini-prep Glycerol stock Archiving Expression
Screening
Purification of Cut PCR Product
RE digestion
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PCR - Set up usingBioRobot 8000
Drosophila Targets
Product obtained with Correct size
A1 - B12

• Bacillus subtillis (30) 95
• Aquifex aeolicus (20) 91
• Archea (60) 93
• Drosophila melanogaster (60) 80

C1 - D12
Drosophila Gene Collection (DGC 1.0) Rubin et
al., Science 2000
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Concentrating Inserts
Lyophilization

• Concentration of inserts (cleaved, purified PCR
  products of the ORFS) is critical for ligation
  process.
• - Low conc. from Qiaquick purification
• - Possible buffer effects (high pH for elution)
• Manual cloning - concentration by ethanol
  precipitation greatly improved ligation
  efficiency
• - concentration
• - further purification
• Tried volatile buffer to elute from Qiaquick
• - poor results.

Resuspend at high concentration in H2O
Desalt by Size Exclusion using 96-well Big Dye
removal plates
Low volume, salt free, high concentration DNA
inserts