Medicinal Structural Genomics of Parasitic Protozoa MSGPP

1
Medicinal Structural Genomics of Parasitic
Protozoa (MSGPP) Protein Production Pipeline
Alberto J. Napuli, Ph.D. MSGPP Protein
Production Unit University of Washington,
Biochemistry Seattle, WA
2

• Medicinal Structural Genomics of Parasitic
  Protozoa (MSGPP)
• April 2006 NIH funded project to determine
  crystal structures of medically relevant proteins
  of pathogenic protozoa. Goal is 50 structures
  and 250 ligand bound.
• MSGPP is composed of 5 units
• Target Selection
• Protein Expression and Purification
• Crystal Growth Unit
• Structure Determination
• Ligand Screening
• Protein Expression and Purification
• Cloning, Screening and Protein Purification
• Recombinant protein expression in E.coli
• Ligation Independent Cloning
• Auto-induction
• 4 purifications per week (200/year)

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Expression vectors
BG1861
BG1861
AVA0421 Cloning Technique LIC LIC Antibiotic
Resistance Amp Amp Promoter T7 T7 N-Terminal
Fusion His His-3C Termination Codon TAA TAA
4
LIC Ready Vector (AVA0421)
3C site
NruI
PmeI
atg gct cat cac cat cac cat cat atg ggt acc ctg
gaa gct cag acc cag ggt cct ggt tcg cga ata ttc
cta gct ttg ttt aaa cag cac gaa caa gtt tac cga
gta gtg gtc gtc gtc gtc tac cca tgg gac ctt cga
gtc tgg gtc cca gga cca agc gct tat aag gat cga
aac aaa ttt gtc gtg ctt gtt caa
RESTRICTION REACTION
3C cleavage site
atg gct cat cac cat cac cat cat atg ggt acc ctg
gaa gct cag acc cag ggt cct ggt tcg tac cga gta
gtg gtc gtc gtc gtc tac cca tgg gac ctt cga gtc
tgg gtc cca gga cca agc
aaa cag cac gaa caa gtt ttt gtc gtg ctt gtt caa
T4 DNA polymerase activity
atg gct cat cac cat cac cat cat atg ggt acc ctg
gaa gct cag acc cA tac cga gta gtg gtc gtc gtc
gtc tac cca tgg gac ctt cga gtc tgg gtc cca gga
cca agc
dATP only
5
aaa cag cac gaa caa gtt Aa
3 to 5 exonuclease activity
PCR Insert
g ggt cct ggt tcg atg-orf-xxxxxxxxxxxxxxxxxx-T
Tag-orf-xxxxxxxxxxxxxxxxxx-a ttt
gtc gtg ctt gtt c
g ggt cct ggt tcg ATG-ORF-xxxxxxxxxxxxxxxxxx-TAA
TAA cag cac gaa caa g c cca gga cca agc
tac-orf-xxxxxxxxxxxxxxxxxx-att att gtc gtg ctt
gtt c
dTTP only
T4 DNA polymerase
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PCR and Ligation Independent Cloning
DNA templates Genomic and cDNA
PCR Gel Extraction Prep
BG1861 AVA0421 LIC Ready Exp. Vectors
REV Primers
T4 DNAP Exonuclease Reaction
FWD Primers
PCR Master Mix
Ligation Independent Cloning LIC
Transformation
NovaBlue
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Colony PCR
NovaBlue Colonies X2
H2O
Master Plate
LB-AMP 37C 16 hr
H2O
REV Primers
FWD Primers
PCR Master Mix
Plasmid Prep
Plasmid DNA Clones
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HT Screens
3 ml 20C PA0.5G 16 hr
3 ml 20C ZY-5052 16 hr
Transformation
BL21(DE3) 37C LB-AMP 16 hr
Plasmid DNA
Glycerol Stock
Room Temp 40 min
Lyses Buffer w/ 0.5 CHAPS Benzonase
-80C
Total/Expression
Wash x3
Ni2 Beads
Elution Buffer 500 mM Imidazole
Soluble Fraction
Wash Buffer 30 mM Imidazole
Waste
Pure/Soluble
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HT Screens-Analyses
Total/Expression
Pure/Soluble
SDS/PAGE Samples 90C / 5 min
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Large Scale Expressions - Upscales
1 ml
0.5 ml
0.1 ml
1000 ml 20C ZY5052 200 rpm 48-72 hr
1 ml Glycerol Stock PA0.5G
AIR vs Shaking
Glycerol Stocks Vs Fresh Colonies
PA0.5G vs LB
Plasmid DNA
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Large Scale Expression
Innova 44R x6 Capacity 6X1L ea / 36L total 2L per
upscale 18 targets per week
LEX 48 Bioreactor x1 Capacity 48L total 48X1L or
24X2L cultures 2L per upscale 24 targets per
week
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Large Scale Expressions - Harvest
1L X2
10 ml
1 ml
Sequencing
10 ml Screen
20-30 Grams -80C
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Purification Schema
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Large Scale Lyses
Lyses Buffer Lysozyme 25 ml
30C water Bath 15 min
1 hr 14,000 rpm 4C
20 mM HEPES pH 7.0 30 mM Imidazole 500 mM
NaCl 0.5 CHAPS 1 mM AEBSF 5 glycerol 1 mM
TCEP Lysozyme
X8
Clarified Sample
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Ni2 Chromatography
Explorer 100 X2
Ni2
Column 5 ml HisTrap FF Equilibration/Wash
Buffer 20 mM HEPES pH 7.0, 500 mM NaCl,
30 mM imidazole, 5 glycerol, 1 mM
TCEP Elution Buffer 250 mM imidazole. Sample
Volume 200-250 ml Sample Pump Flow Rate 5
ml/min System Pump Flow Rate 5
ml/min Equilibration 10 CV Wash 20
CV Elute 10 CV Fraction Size 5 ml Total
Fractions 12
15
3C-protease cleavage reaction
3C Protease 150
20 mM HEPES pH 7.0 60 mM Imidazole 800 mM
NaCl 5 glycerol
Dialyses 4C/16 hr
20 mM HEPES pH 7.0 30 mM Imidazole 500 mM
NaCl 5 glycerol
Concentration 3-20 mg/ml Total protein 30-200
mg Volume 6-20 ml
20 mM HEPES pH 7.0 200 mM NaCl 5 glycerol 1
mM DTT
20 mM HEPES pH 7.0 250 mM Imidazole 500 mM
NaCl 5 glycerol 1 mM TCEP
Ni2
Ni2
FT 15-40 ml
W 10 ml
E 10 ml
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SEC Chromatography
Column 300 ml 26/60 Superdex 75 SEC
Buffer 20 mM HEPES pH 7.0, 500 mM NaCl,
5 glycerol, 1 mM TCEP Sample Volume
6-12 ml Sample Pump Flow Rate 0.5-1
ml/min System Pump Flow Rate 0.5-1
ml/min Equilibration 0.5 CV Elution 0.7
CV Fraction Size 5 ml Total Fractions 47
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HPLC system and column maintenance
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Protein Concentration
20 to 2 ml
concentrate
Pre-Crystalyzation Test PCT
Dilute
96 Well PCR Plate 20-50 ul / well
Flash Freeze
Store at -80C
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Acknowledgements
Wim Hol Wesley Van Voorhis Christophe
Verlinde Fred Buckner Ethan Merritt Erkang
Fan Structure Determination Unit Isolde Le
Trong Jüergen Bosch Jon Caruthers Eric
Larson Crystallization Growth Unit Brian
Krumm Jenni Ross Helen Neely Liren Xiao Li
Zhang
Protein, Expression Purification Alberto
Napuli Angela M Kelley Natascha Mueller
Lisa Castaneda Soraya Aalami Selma Alkafeef
Hauptman-Woodward MRI George DeTitta Joseph
Luft Angela Lauricella Ligand Synthesis
Screening Sayaka Shibata Zhongsheng Zhang
Informatics and Databases Frank Zucker
Christine Stewart
Supported by NIAID.