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