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

Expanding Parallel Drug Discovery into Gene Families

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

pBEV: Dual-System Expression Vector

(Chambers et al 2004)

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)

Automated baculo-viral production

qPCR used to determine viral titer

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)

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.biotech.ou.edu/)(http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/cai.html)

Effect of Protein Size on Expression & Solubility in E.coli

Quantitative Analysis of Expression Strategies:Decision Tree Analysis of Kinase Expression

86% Structures

14% Structures

Actual Payoff in Kinase Structures

Novel Kinase Structures in PDB

(Yon & Jhoti 2003)

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 MAP Kappa 2 Kinase

(Meng et al 2002)

MAPK2K∆46

MAPK2K

MAPK2K ∆46 2.8 Å

Crystal Structure of Aurora-2 Kinase

Limited Proteolysis

(Cheetham et al 2003)

Aurora2K ∆107 2.9 Å

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    

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

MiniaturizedAutomated

CrystallizationMultiple 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

Molecular CloningProtein Expression

Protein Biochemistry

X-Ray Crystallography

Enzymology