protein production for structure-based drug design stephen chambers ~ head of gene expression vertex...
TRANSCRIPT
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
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
Content
• Structural Genomics vs Structure Based Drug Design – Much in common
• Expression strategy for higher output– 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
Cloning
Expression
Purification
Crystallography
DNA
Enzymology
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:
• Expression systems• Growth conditions• Cell lines• Constructs• Mutants
Expression Systems
E.COLI YEAST INSECTCELLS
MAMMALIANCELLS
ProteolyticCleavage
+/- +/- + +
Glycosylation - + + +
Secretion +/- + + +
Folding +/- +/- + +
Phosphorylation - + + +
Yield (%)based on dry wt
1-5 1 30 <1
(Valk & Keus 1990)
Wave Reactor
(Wave Biotech)
HiGro Shaker (Genomic Solutions Inc)
Highly Engineered Process Using Standard Equipment
Ni-NTA Magnetic
Agarose Beads
Genesis (Tecan)
Cup-horn sonicator
(Misonix Inc)
Model Behavior in E.coli
(http://www.biotech.ou.edu/)(http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/cai.html)
Quantitative Analysis of Expression Strategies:Decision Tree Analysis of Kinase Expression
86% Structures
14% Structures
Actual Payoff in Kinase Structures
HT-Expression Allows the Exploration of Diversity: Rapid Identification of Well Expressing Proteins
Pim-1
2.4 Å
GSK3
2.7 Å
(ter Haar et al 2001)
Crystal Structure of FLT-3 Kinase
(Griffith et al 2004)
FLT-3(H564-S993) 2.1 Å
DOMAIN DELETION DUPLICATION MUTANTH564-S993 ITD H564-S993 H564-S993 H711-V782 H564-S993 F723-H761 H564-V958 ITD H564-V958 H564-V958 D835YH564-V958 H711-V782 H564-V958 H711-H761 H564-V958 F723-V782 H564-V958 F723-H761 H564-F936 H564-F936 H711-V782 H564-F936 F723-H761 N587-S993 ITD N587-S993 N587-S993 H711-V782 N587-S993 F723-H761 N587-V958 ITD N587-V958 N587-V958 H711-V782 N587-V958 H711-H761 N587-V958 F723-V782 N587-V958 F723-H761 N587-F936 ITD N587-F936 N587-F936 H711-V782 N587-F936 F723-H761 W603-S993 W603-S993 H711-V782 W603-S993 F723-H761 W603-V958 W603-V958 H711-V782 W603-V958 F723-H761 W603-F936
Expressing Difficult Proteins in Insect Cells: Proteases
Cathepsins Serine proteases Metallo proteases
Integrated Platform Serving Structural Biology
MiniaturizedAutomated
CrystallizationMultiple Inhibitor
Structures for Drug Design
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
Molecular CloningProtein Expression
Protein Biochemistry
X-Ray Crystallography
Enzymology