Getting the Most Out of Baculovirus Linda Lua

Enabling World Class Research

•  Recombinant Protein Production ▫  Discovery, Translational, Preclinical ▫  Drug discovery, vaccinology, diagnostics, functional

materials, targeting and delivery, therapeutics, bioprocess development, agricultural science

•  Research Training ▫  Up-skill professional development ▫  Tailored workshops and symposium

•  Consultation

Design & HTP Molecular Cloning

HTP Expression Screening

Scale-up Expression

Protein Purification & Analysis

Verified Construct

“A focus of expertise in protein research and a research emphasis on platform technologies”

Which Way Should I Go?

Types of Recombinant Protein

• Enzymes • Glycoproteins • Cytokines/interleukins • Kinases •  Transcription factors •  Structural proteins • Membrane proteins • Receptors • Viral proteins • Recombinant peptides

http://www.rsc.org/ej/NP/2010/c0np00005a/c0np00005a-f4.gif http://nfs.unipv.it/nfs/minf/dispense/immunology/lectures/files/structure_abs_tcr.html

Army Malaria Institute

International Academic & Industry

Australia

•  Brazil •  Germany •  New Caledonia •  New Zealand •  North America •  Malaysia •  Singapore •  United Kingdom

•  16 Australian universities •  11 Government research

organizations •  13 Companies

www.uq.edu.au/pef

•  >1000s expression constructs

•  >100s proteins •  Simple biomolecules to

complex multi-protein assemblies

Baculovirus Technology 1980s •  Occasional production of

‘difficult to express’ proteins

•  Rescue projects

2014 •  A major expression tool in

most pharma/biotech labs •  50-60% proteins expressed

using baculo •  Vaccines and gene delivery

system

Technology enhancement Automation

High-throughput

COST%

SPEED%

TYPICAL%YIELD%

POST%–%TRANSLATION%MODIFICATION%

FDA%APPROVAL%

LOW% HIGH%

BACULO8INSECT%CELL% CELL8FREE%BACTERIA%MAMMALIAN% YEAST%

BACULO8INSECT%CELL%CELL8FREE%BACTERIA% MAMMALIAN%YEAST%

BACULO8INSECT%CELL%CELL8FREE% BACTERIA% MAMMALIAN%YEAST%

BACULO8INSECT%CELL%CELL8FREE% BACTERIA%MAMMALIAN% YEAST%

BACULO8INSECT%CELL%CELL8FREE% BACTERIA% MAMMALIAN%YEAST%

rBaculovirus

H1, H5, H10 wild-type & mutants

Influenza VLP

HPV VLP

chimeric VLP

•  Dengue viral proteins •  Malaria proteins •  TB proteins •  Hendra viral proteins •  Receptors and ligands •  Ion channels •  Kinases •  Glycoproteins •  Enzymes •  Interleukins

Virus-like particles Hemagglutinin

100s recombinant proteins produced in this system

HTP Baculovirus/Insect Cell Platform Recombination

24-well Virus amplification

24-deep well

Expression screening 24-deep well

Day 0 Day 7

Day 10

Expression analysis

Day 13

Cloning 96-well

1 week

Common Challenges for Secreted Proteins

Low expression/secretion

Large volume of supernatant

Poor target protein recovery and purity

Contaminating host proteins

Media: Sf900II P3 virus stock MOI 5 TOI hi5-1.5x106 cells/ml TOI Sf9-3x106 cells/ml

27oC

21oC

FlashBACGOLD™ Bac to Bac™

HBM HBM gp67 gp67

High Five™ gp67 Bac to Bac™ - 21oC FlashBACGOLD™ Δv-cath ΔchiA- 27oC

Ni Sepharose™ Excel Bind-Elute

Stability of Virus Stock

P3 P4 P5 P6 P7 P8

Virus-Like Particle (VLP)

Simple to Complex Virus-Like Particles

•  Single layer •  Multiple layers •  Non-enveloped •  Enveloped

Expert Rev. Vaccines 9(10), 1149–1176 (2010)

Enteroviruses

• Hand Foot and Mouth Disease (HFMD) •  Poliomyelitis

Enterovirus Genome and Viral Structure

Enterovirus-like Particles as Vaccines •  Virus-like particle (VLP) resemble virus •  Non-infectious as without genetic

material •  Produce using insect cells •  Co-expression of polyprotein (P1) and

protease (for processing of polyprotein into structural proteins)

•  Assembly of structural proteins in VLPs in cells

Flow fractionation (AF4)

Membrane Crossflow

MALS, UV & RI

size

%

Size Distribution Velocity

1. Sample enters chamber.

Process:

2. Crossflow drags particles to membrane 3. Particles diffuse back into flow (rate based on size).

4. Smaller particles flow faster than larger particles due to laminar flow.

5. UV, MALS & RI used to determine size distribution

Analysis by field-flow fractionation

VLPs

Aggregates

Misformed

VLPs

VLPs

Misformed VLPs

Aggregates

Chuan et al., Biotech Bioeng 2008

Biophysical Characterization

DLS analysis: Diameter - 32 nm TEM analysis: Diameter – 25-30 nm

AF4 analysis: Diameter - 30 nm

0"

10"

20"

30"

40"

50"

60"

70"

80"

90"

100"

110"

0"

0.2"

0.4"

0.6"

0.8"

1"

1.2"

0" 10" 20" 30" 40" 50" 60"

Radius'(n

m)'

UV'absorba

nce'(rela4

ve'scale)'

Time'(min)'

Soluble""Proteins"

VLPs"

Aggregates"

Misformed""VLPs"

28

Modular VLP Vaccine Design

Virus-like particle

VP1 Modular VP1 Modular capsomere (5 VP1)

Modular VLP (72 capsomeres)

Modular VLP Vaccine Design

Modular VP1

1.  Correct epitope presentation is crucial to induce immune response against target pathogen

2.  Module insertion must not disrupt capsomere structure

Computational tools to design and predict modular vaccine

candidates, test using insect cells

29

30

Modular VLP Vaccine Design

Virus-like particle

VP1 Modular VP1 Modular capsomere (5 VP1)

Modular VLP (72 capsomeres)

The Future…

FLU

“A focus of expertise in protein research and

a research emphasis on platform technologies”

Our track record Our expertise Our capabilities Our platform

Chancellor’s Award for Team Excellence 2013

THANK YOU