high-throughput protein production platform for the northeast structural genomics consortium er82...
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High-Throughput Protein Production Platform for the Northeast Structural Genomics
Consortium
ER82 WR66
Thomas Acton, Ken Conover, Bonnie Cooper, Yiwen Chiang, Natalia Denissova, Chi Kent Ho, Li-Chung Ma, Ritu Shastry, Lydia Shih, Rong Xiao, Stephen Anderson, Masyori Inouye,
Gaetano T. Montelione
Rutgers Protein Production:
600 Soluble Proteins Yearper week per year
Clones / Solubility Testing 50 2500
12/50 (25%) 600Large Scale Ferm/Purification
2/12 (15%)Crystal Hits 100
3D Crystal Structures 1/12 (~10%) 50-60
“Good” HSQC 3/12 (25%) 150
3D NMR Structures >1/12 (~10%) >50-60
Rost Clusters: Structural Genomics Targets
• Protein domain families / clusters
• Full length proteins < 340 amino acids
• No member > 30% identity to PDB structures
• No regions of low complexity
• Not predicted to be membrane associated
Target genomes Reagent genomes (prokaryotes):Eukaryotes Eubacteria Archea
Arabidopsis thaliana (A) Aquifex aeolicus (Q) Aeropyrum pernix (X)Caenorhabditis elegans (W) Bacillus subtilis (S) Archaeoglobus fulgidus (G)Drosophila melanogaster (F) Escherichia coli (E) Methanobacterium thermoautotrophicum (T)
Homo sapiens (H) Haemophilus influenzae (I) Pyrococcus horikoshii (J)Saccharomyces cerevisiae (Y) Helicobacter pylori (P) Pyrococcus furiosus (PfR)
Staphylococcus aureus (Z)Thermotoga maritima (V)Campylobacter jejuni (B)
Neisseria meningitides (M)Thermus thermophilus (U)
etc
~ 20,000 Rost Clusters
Aeropyrum pernixAquifex aeolicusArabidopsis thalianaArchaeglobus fulgidisBacillus subtilisBrucella melitensisCaenorhabditis elegansCampylobacter jejuniCaulobacter crescentus
Deinococcus radioduransDrosophila melanogaster
Escherichia coliFusobacterium nucleatumHaemophilus influenzaeHelicobacter pyloriHomo sapiens
Human cytomegalovirus Lactococcus lactisM. thermoautotrophicumNeisseria meningitidisOtherPyrococcus furiosusPyrococcus horikoshi
Saccharomyces cerevisiaeStaphylococcus aureusStreptococcus pyogenesStreptomyces coelicolor
Thermoplasma acidophilumThermotoga maritimaThermus thermophilusVibrio cholerae
Phylogenetic Distribution of NESG Target Proteins
Homo sapiens
S. cerevisiae
C. elegansD
. melanogaster
Arabidopsis thaliana
Intra and Inter-Laboratory Coordination is Maintained by the SPINE Database
http://spine.nesg.org/sum.pl
AR81 HSQC
Multiplex Expression System
Classical Restriction Endonuclease/Ligase-
dependent cloning
E. coli Expression Vectors
Eukaryotic Expression systems
96 Well Primer Design
http://www-nmr.cabm.rutgers.edu/bioinformatics/index.html
Everett et al., 2004. J Struct Funct Genomics. In Press
DNA Mini-preps PCR ReactionSet up-96 well
PCR Gel Puri.
Res
tric
tio
n D
iges
t
Qiaquick Purify
Lig
atio
nT
ran
sfo
rm C
olo
ny
PC
R
Cycle Sequencing
Big Dye removal
Auto-Steps with the Biorobot 8000
cDNA Synthesis / RT-PCRBottleneck for cloning Eukaryotic genes
cDNA-Template for PCRcDNA Libraries -
1. Availability - Cost etc.
2. Not fully sequenced - Quality not fully spliced/length, etc.
3. Transfer - 384 -Well plates
4. Representative - not all tissue/development etc.
A. Extract RNA (total)/PolyA
B. Reverse Transcription from mRNA
Oligo dT primer
Common template for all reactions Similar to Bacterial cloning
PCR of Fly and Human Targets
• PolyT priming of PolyA RNA Drosophila embryo, larva & adult
• Homo sapiens, adult brain adenocarcinoma, & testes.
E. coli PCR
Organism Correctsize
# oftargets
E. coli 99% 120Bacillus subtillis 95% 141Archea 93% 116Aquifex aeolicus 91% 96D. melanogaster* 87% 151H. sapiens* >70% 596*RT-PCR
Primer Prim’_r Success RateHomo sapiens RT-PCR
561462
462459
462462
462657
417207
681390
100
bp
HR
556
HR
558
HR
559
HR
560
HR
561
HR
562
HR
563
HR
564
HR
566
HR
569
HR
570
HR
565
bp
Plate Reader
Transformation
Har
vest
Pla
te C
entr
ifu
ge
Well Abs ug/mlA6 0.477 137B8 0.516 152C7 0.279 60E6 0.771 251E9 0.37 95F4 0.359 91G4 0.66 208D10 0.092 <1B4 0.201 30
ConcentrationOvernight Culture
96 Well Transfer (MJ9)
96-Well Culture
IPTG
24-well blocks96 to 24 Well Transfer
O/N
@17
o C
96-Well Ni-NTA
Purified Protein
A6 B8 C7 E6 E9 F4 G4D10 B4
SDS-PAGEInduction
37o C
96-Well Plate
24-Well Block
2.2 ml S-Block
2.2 ml S-Block37o C
37o C
BL21
LB
96-Well Plate
96-Well Plate
96-Well Plate
Tra
nsf
er P
rote
in
Bra
dfo
rd-9
6 W
elll
96-Well Expression ScreeningPlate Sonicate
Cloning/Expression Summary2 colonies % Cloned/Correct PCR Product
E. coli 91
B. subtilis 98
D. melanogaster 85
H. sapiens 93803915
1064113612081258130813621432149115601655174718381934209821942394
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
Jan '02Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Jan '03Feb Mar Apr May Jun Jul AugSept Oct Nov Dec
Jan '04Feb Mar
Clones vs Time
Automation
0
100
200
300
400
500
600
700
800
900
1000
Jan '03Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Jan '04Feb Mar
Fermentation and Purification
17o C Room 55 - 2L Positions
Akta 3-D
Ferm/Purify 0.5 L N15-MJ1.0 L SeMet1L 13C,15N
Step 1: Ni-Affinity Chromatography
Taylor Graham ‘04
HR969
HSQC ScreeningAmenability to Structural Determination by NMR
# Targets Good Excellent314 60 25
20% 8%
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Sample Optimization - Buffer Screening
Microdialysis Buttons- Optimization for NMR
Buffer NaCl DTT Arginine
50 mM Ammonium Acetate pH 5.0 0 0 0
50 mM Ammonium Acetate pH 5.0 0 10 mM 0
50 mM Ammonium Acetate pH 5.0 0.1 M 10 mM 0
50 mM Ammonium Acetate pH 5.0 0 10 mM 0.1 M
50mM MES pH 6.0 0 0 0
50mM MES pH 6.0 0 10 mM 0
50mM MES pH 6.0 0.1 M 10 mM 0
50mM MES pH 6.0 0 10 mM 0.1 M
50mM Bis.Tris pH 6.5 0 0 0
50mM Bis.Tris pH 6.5 0 10 mM 0
50mM Bis.Tris pH 6.5 0.1 M 10 mM 0
50mM Bis.Tris pH 6.5 0 10 mM 0.1 M
Vary Buffer Conditions - Stability
Screen for ppt.
100 mM Arginine Small sample mass
(50 ug/button)
Bagby S, Tong KI, Liu D, Alattia JR, Ikura M. 1997. J Biomol NMR.
Monodisperse Conditions
Aggregation Screening - Crystallization
Analytical Gel Filtration with Light Scattering
Proterion - 96 Well
Less Sample
More Conditions
Philip Manor, Roland Satterwhite and John Hunt
LS
RI
Gel Filtration Under Monodisperse Conditions
Explorer PurifierFour Akta Primes
•Pool Fractions
•Concentrate (~10 mg/ml)
•Small Aliquots
•Flash Freeze
•Ship to Columbia/HWI
Superdex 75
5 hours
12 hours
ÄKTAxpress™
4 modules in parallel 16 samples AC-GF/DS
AC
AC/GF
Affinity Chromatography (AC)HiTrap™ Chelating HP, 1 and 5 ml
Gel Filtration (GF)HiLoad 16/60 Superdex 200 pg
Distribution of the First 99NESG Structures
Phylogenetic
All targets are members of eukaryotic protein families
1
2NMRX-ray
MethodEuk
aryo
tic
Eubacteria
Archea
Solubility/2004 Stats
Organism Cloned % Sol* PDBA. aeolicus (Q) 85 46 3A. thaliana (A) 35 29 1A. fulgidis (G) 23 74 2B. subtilis (S) 158 49 4B. melitensis (L) 15 67 0C. elegans (W) 90 50 6C. jejuni (B) 20 55 0D. melanogaster (F) 113 15 1E. faecalis (Ef) 23 100 0E. coli ( E) 118 50 12H. influenzae (I) 101 57 4H. pylori (P) 75 21 1H. sapiens (H) 548 43 4N. meningitidis (M) 22 54 1P. furiosus (Pf) 48 46 2P. horikosh i (J) 19 63 1S. pyogenes (D) 12 50 1
*defined as greater than 60% soluble by SDS-PAGE analysis
•8 HR (Human) proteins in advanced stages of NMR
•3 HR Crystal structures
Total Week GoalCloned 511 51 50Fermented 183 20 ~20-24Purified 180 20 ~20-24
2004 ProductionSolubility vs Organism
2004 HR Success
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