Transcript Document

Protein Production for Structure-Based
Drug Design
Stephen Chambers
~
Head of Gene Expression
Vertex Pharmaceuticals Incorporated
NIGMS 2004 PSI Protein Production & Crystallization Workshop
March 29-31, 2004
Vertex: Building a Major Drug Company
Focus:
Small molecule drugs for major diseases
• Established: 1989; Public: 1991
• Common stock: NASDAQ: VRTX
• >700 employees
• 3 sites
– Cambridge, US (headquarters)
– Oxford, UK
– San Diego, CA
Traditional vs. Vertex Approach to Drug Discovery
Therapeutic Area Perspective
• Single target approach
Target Family Perspective
• Chemogenomics multi-target approach
Expanding Parallel Drug Discovery into
Gene Families
Content
• Structural Genomics vs Structure Based Drug Design
– Much in common
• Expression strategy for higher output
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–
–
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Parallel expression of E.coli & insect cells
Quantitative analysis
Illustrated using examples from Vertex Kinase program
Application to other protein families
• Integration into a broader process with other
disciplines
Structure Based Drug Design
• Drug Discovery (& Development)
• Human proteins
– Complex post-translationally modified proteins
– Heterogeneous proteins
– ‘Difficult’ proteins
• Highest value structures contain inhibitor
• Premium given to high-output (cf high-throughput)
• Failure not an option
Protein Expression Bottleneck
DNA
Cloning
Expression
Purification
Enzymology
Crystallography
Consumers:
• Protein Biochemistry
– soluble, purifiable protein
• Enzymology
– soluble, active protein
– 0.1-10 mg of protein
• Crystallography
– soluble, crystallizable protein
– 5-100 mg of protein
Expression Process
Triage Expression
Prior to Production:
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Expression systems
Growth conditions
Cell lines
Constructs
Mutants
pBEV: Dual-System Expression Vector
(Chambers et al 2004)
Expression Systems
E.COLI
YEAST
INSECT
CELL S
MAMMALIAN
CELL S
Proteolytic
Cleavage
Glycosylation
+/-
+/-
+
+
-
+
+
+
Secretion
+/-
+
+
+
Folding
+/-
+/-
+
+
Phosphorylation
-
+
+
+
Yield (%)
based on dry wt
1-5
1
30
<1
(Valk & Keus 1990)
Automated baculo-viral production
qPCR used to determine viral titer
Highly Engineered Process Using Standard Equipment
HiGro Shaker (Genomic Solutions
Inc)
Genesis (Tecan)
Ni-NTA Magnetic
Agarose Beads
Wave Reactor
(Wave Biotech)
Cup-horn
sonicator (Misonix
Inc)
HT-Expression & Automated Purification
Full-Length Kinases in E.coli & Insect cells
Increasing solubility in E.coli:
TK>AGC>STE>>CMGC>CAMK
Model Behavior in E.coli
(http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/cai.html)
(http://www.biotech.ou.edu/)
Effect of Protein Size on Expression & Solubility in E.coli
Quantitative Analysis of Expression Strategies:
Decision Tree Analysis of Kinase Expression
Actual Payoff
in Kinase Structures
14% Structures
86% Structures
Novel Kinase Structures in PDB
(Yon & Jhoti 2003)
HT-Expression Allows the Exploration of Diversity:
Rapid Identification of Well Expressing Proteins
GSK3
2.7 Å
(ter Haar et al 2001)
Pim-1
2.4 Å
Crystal Structure of MAP Kappa 2 Kinase
MAPK2K ∆46
2.8 Å
MAPK2K
MAPK2K∆46
(Meng et al 2002)
Crystal Structure of Aurora-2 Kinase
Limited Proteolysis
Aurora2K ∆107
2.9 Å
(Cheetham et al 2003)
Crystal Structure of FLT-3 Kinase
FLT-3(H564-S993)
2.1 Å
DO MAIN
H564-S993
H564-S993
H564-S993
H564-S993
H564-V958
H564-V958
H564-V958
H564-V958
H564-V958
H564-V958
H564-V958
H564-F936
H564-F936
H564-F936
N587-S993
N587-S993
N587-S993
N587-S993
N587-V958
N587-V958
N587-V958
N587-V958
N587-V958
N587-V958
N587-F93 6
N587-F93 6
N587-F93 6
N587-F93 6
W603-S 993
W603-S 993
W603-S 993
W603-V 958
W603-V 958
W603-V 958
W603-F936
DELET ION
DUPLICATI ON MUTA NT
ITD
H711-V782
F723-H761
ITD
D835Y
H711-V782
H711-H761
F723-V782
F723-H761
H711-V782
F723-H761
ITD
H711-V782
F723-H761
ITD
H711-V782
H711-H761
F723-V782
F723-H761
ITD
H711-V782
F723-H761
H711-V782
F723-H761
H711-V782
F723-H761
(Griffith et al 2004)
HT-Expression Allows the Exploration of Diversity:
Mutations
HT-Expression Allows the Exploration of Diversity:
Utilizing Different Cell Lines
Insect Cell Expressed Structures in PDB
E.coli Expressed Structures in PDB
From the Vertex Portfolio
Source of Proteins
Source of Structures
Expressing Difficult Proteins in Insect Cells: Proteases
Cathepsins
Serine proteases
Metallo proteases
HT-Expression Allows the Exploration of Diversity:
Protein Families (Phosphatases)
HT-Cloning & Expression Process
HT-Purification & Crystallography Process
No Protein Left Behind: Rescuing Insoluble Proteins
CDC25a
1.7 Å
ICE
2.6 Å
No Protein Left Behind: Rescuing Soluble Proteins
Integrated Platform Serving Structural Biology
Miniaturized
Automated
Crystallization
Multiple Inhibitor
Structures for Drug Design
Novel Kinase Structures: Not as Interesting as the Active Site
An Active Site with Various Inhibitors
Conclusions
• Demonstrated efficient protein production integrated into a platform for
structure-based drug-design
• Insect cells expression negates many of the deficiencies observed in
E.coli expression
• High-throughput expression used to identify soluble expressed protein
– proteins that are difficult to express and insoluble are usually difficult to
purify and crystallize
• Parallel expression in E.coli and insect cells, providing greater number
of soluble expressed protein, increases operational efficiency
• Process applicable to a range of gene families
Acknowledgements
Protein Expression
Protein Biochemistry
Molecular Cloning
Enzymology
X-Ray Crystallography