BEVS Screen for Improved Protein Survival · Recombinant Protein Production Discovery, Translational, Preclinical Drug discovery, vaccinology, diagnostics, functional materials, targeting

BEVS Screen for Improved Protein Survival Cindy Chang

Enabling World Class Research

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

functional materials, targeting and delivery, therapeutics, bioprocess development

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

• Consultation

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

Vorführender

Präsentationsnotizen

Brazil – Brazilian centre for research in energy and materials, Germany – Lead discovery centre, New Caledonia – Institute Agronomic New Caledonia, New Zealand – Univeristy of Canterbury, North America – McGill University, Malaysa – Sentinext, Singapore – Nanyang Technological University, UK – Pirbright Insitute

Design & HT Molecular Cloning

HT Expression Screening

Scale-up Expression

Protein Purification & Analysis

Verified Construct

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

Benchmarking Baculovirus- Insect Cell Expression System

Goal

• Compare the performance of the BEVS setup within participating facilities ▫ In-house routine protocol

• What can we learn from one another?

• Any particular trends in the results?

Characteristics

Virus Specifications Modifications

Bac-to-Bac®

• Viral genome as BAC • Blue-white screening

No modifications

MultiBacTM • Viral genome as BAC • Blue-white screening • Multi-gene cassettes integrated into the

same baculovirus for protein complexes

ΔchiA, Δv-cath

EmBacY • Viral genome as BAC • Blue-white screening • YFP reporter gene in virus backbone

ΔchiA, Δv-cath

BEVS

Vorführender


B2B is based on site specific transposition to use transposon to insert GOI into bacmid DNA propagated in E. Coli. Colonies containing recombinant bacmid identified by antibiotic and blue/white selection as it disrupts the lacz gene. Based on the need to generate recombinant bacmids. The viral genome is maintained in E. coli cells as a bacterial artificial chromosome. Extended cell viability Reduced proteolysis YFP reporter to monitor virus performance and protein production in real time

BEVS Characteristics

Virus Specifications Modifications

BaculoDirectTM • Gateway® Technology • In vitro recombination with linear

baculovirus

No modifications

flashBACTM

• Defective viral genome • One-step co-transfection allows

homologous recombination within cells & generates recombinant virus

ΔchiA

flashBACGOLDTM

ΔchiA, Δv-cath

flashBACULTRATM

ΔchiA, Δv-cath Δp10, Δp74, Δp26

flashBACPrimeTM

No modifications

BacMagicTM

• Defective viral genome • One-step co-transfection allows

homologous recombination within cells & generates recombinant virus

ΔchiA

Vorführender


Baculodirect uses linearised baculovirus for in vitro homologous recombination FB uses homologous recombination. Baculovirus missing essential portion of the genome for replication P10, p74 and p26 are non-essential genes for virus replication. Deletion removes competitor for limiting cellular resources.

BEVS Pipeline

0 5 10 15 20

BacMagic™

flashBAC™

BaculoDirect™

EmBacY

MultiBac™

Bac-to-Bac®

Days

Bacmid PreparationTransfectionVirus AmplificationExpression Screening

Vorführender


Each of the systems has its own advantages and disadvantages. One important factor to consider is time. EmBacY, MultiBac, Bac-to-Bac: 18 days BaculoDirect, flashBAC: 13 days

• Cell line • Media • Temperature • Vessel

• MOI • TOI • TOH

• Baculovirus system

• Genome modifications

• Promoters • Signal peptide

Vector Design BEVS

Culture Conditions

Infection Strategies

Protein Candidates

• 4 Intracellular ▫ PEPF-Muc: 203.9 kDa ▫ cAbl1wt: 126.4 kDa ▫ FMRP: 69.9 kDa ▫ NS1-H1: 78.4 kDa

• 2 Secreted ▫ CSF1: 18.9 kDa ▫ soluble G protein: 60.9 kDa

Vorführender


These are the 6 protein candidates that has been selected based on difference in protein size, yield and localisation

HTP Baculovirus/Insect Cell Platform Transfection

24-well Virus amplification

24-deep well

Expression screening 24-deep well

Expression analysis

Cloning 96-well

1 week Day 0 Day 7

Day 10 Day 13

Bac-to-Bac® flashBACULTRATM

ΔchiA, Δv-cath, Δp10, Δp74, Δp26

Our BEVS Set-Up

Vorführender


Our standard system is flashBACUltra which

flashBACULTRATM Bac-to-Bac® Intracellular Candidates

pBac1-PEPF-Muc pBac1-cAblwt pBac1-FMRP pBac1-NS1-H1

pFB-PEPF-Muc pFB-cAblwt pFB-FMRP pFB-NS1-H1

Secreted Candidates

pBac1-HBM-CSF1 pBac1-gp67-CSF1 pBac1-HBM-sG pBac1-gp67-sG

pFB-HBM-CSF1 pFB-gp67-CSF1 pFB-HBM-sG pFB-gp67-sG

Molecular Cloning

*16 constructs

Parameters for Intracellular Expression

BEVS

Bac-to-Bac®

flashBACULTRATM (ΔchiA, Δv-cath,

Δp10, Δp74, Δp26)

Cell Line

Sf9

High FiveTM

Temperature

27oC

21oC

Media: Sf900II P3 virus stock MOI 5 TOI Sf9-3x106 cells/mL TOI Hi5-1.5x106 cells/mL

Vorführender


P10, p74 and p26 are non-essential genes for virus replication. Deletion removes competitor for limiting cellular resources. flashBACULTRA™ contains deletions of the chitinase and cathepsin genes, as above, but also deletions of the p10, p74 and p26 genes and provides the best choice for the most difficult to express proteins; suitable for cytoplasmic, nucleus, secreted and membrane proteins.

PEPF-Muc 203.9 kDa

260

160

MW (kD) MW (kD)

27oC

27oC

21oC

21oC

48 72 96 96 120 144 48 72 96 96 120 144

260 160

48 72 96 96 120 144 48 72 96 96 120 144

27oC

27oC

21oC

21oC

flashBACULTRATM Bac-to-Bac®

Sf9 High FiveTM Sf9 High FiveTM

cAb1wt 126.1 kDa

260

160

110

MW (kD) MW (kD)

260

160 110

27oC

27oC

21oC

21oC

48 72 96 96 120 144 48 72 96 96 120 144

27oC

27oC

21oC

21oC

48 72 96 96 120 144 48 72 96 96 120 144

FMRP 70.1 kDa

110

80

60

110

80

60

MW (kD) MW (kD)

27oC

27oC

21oC

21oC

48 72 96 96 120 144 48 72 96 96 120 144

27oC

27oC

21oC

21oC

48 72 96 96 120 144 48 72 96 96 120 144

Intracellular Proteins

NS1-H1 78.4 kDa

MW (kD) MW (kD)

27oC

27oC

21oC

21oC

48 72 96 96 120 144 48 72 96 96 120 144 48 72 96 96 120 144 48 72 96 96 120 144

27oC

27oC

21oC

21oC

110

80

60

110

80

60

Vorführender


Expression screening is critical to maximise protein yields.

Parameters for Secreted Expression

BEVS

Bac-to-Bac®

flashBACULTRATM (ΔchiA, Δv-cath,

Δp10, Δp74, Δp26)

Cell Line

Sf9

High FiveTM

Temperature

27oC

21oC

Signal Peptide

Honeybee melittin (HBM)

Glycoprotein gp67 (gp67)


Vorführender


flashBACGOLD™ is enhanced by the deletion of the chitinase and cathepsin protease genes. It provides superior levels of expression for any protein that is destined for secretion or to be inserted in the membrane, or for any protein that might be particularly liable to degradation.

CSF1 18.9 kDa

24 48 72 96 24 48 72 96

flashBACULTRATM

HBM gp67

72 96 120 144 72 96 120 144

Sf9

High FiveTM

27oC

24 48 72 96 24 48 72 96

Bac-to-Bac®

HBM gp67

72 96 120 144 72 96 120 144

21oC

Sf9

High FiveTM

24 48 72 96 24 48 72 96 24 48 72 96 24 48 72 96

72 96 120 144 72 96 120 144 72 96 120 144 72 96 120 144

Sf9

Sf9

High FiveTM

High FiveTM

27oC

21oC

HA1 37.4 kDa

High FiveTM

21oC

24 48 72 96

72 96 120 144

24 48 72 96 24 48 72 96 24 48 72 96

72 96 120 144 72 96 120 144 72 96 120 144

Sf9

Sf9

High FiveTM

27oC

Soluble G 60.9 kDa

Secreted Glycoproteins

Our observations • The choice of BEVS affects protein expression • Higher expression observed in High FiveTM than Sf9

insect cells • gp67 signal peptide is an effective signal sequence

for improved yields of secreted protein • Lowering the culture temperature has an increased

impact on the Bac-to-Bac® system • Optimise expression conditions is critical for

maximising protein yield

What about complex protein assemblies?

DNA

Virus-like Particles (VLPs)

• Mimicks the organization and conformation of native viruses

• Non-infectious as without

genetic material • Tremendous potential as

vaccine candidates! Infectious virus VLP

Virus-Like Particle Assembly

• Multimeric protein complexes ▫ Single or multiple capsid proteins

• Envelope or non-enveloped VLPs • Assembly of structural proteins into VLPs in

vivo in insect cells

Monomer Intermediate VLP

Parameters for VLP Expression

BEVS

BaculoDirectTM

Bac-to-Bac®

flashBACTM

flashBACGOLDTM

flashBACULTRATM

flashBACPrimeTM

Cell Line

Sf9

High FiveTM

Temperature

27oC

21oC


Vorführender


flashBACPrime combines the simplicity of the flashBAC™ one step baculovirus expression system with increased recovery and yield of virus like particles (VLPs).flashBACPRIME™ is based on the original AcMNPV genome without any of the gene deletions characteristic of the other flashBAC™ range of vectors. Thus cells infected with flashBACPRIME™ induce cell lysis in the late stages of infection. Cell lysis facilitates release and subsequent purification of VLPs (or other proteins) that form in the cytoplasm or nucleus of infected cells.

Single-Capsid VLP

Sf9

48 72 48 72 48 72 48 72 48 72 48 72

High FiveTM

27oC

50

40

MW (kD) MW (kD)

50

40

21oC

50

40 50

40

Bac

uloD

irec

tTM

flash

BA

CTM

flash

BA

CG

OL

DTM

flash

BA

CU

LT

RA

TM

flash

BA

CPR

IME

TM

Bac

-to-

Bac

®

Bac

uloD

irec

tTM

flash

BA

CTM

flash

BA

CG

OL

DTM

flash

BA

CU

LT

RA

TM

flash

BA

CPR

IME

TM

Bac

-to-

Bac

®

VP1 42.5 kDa

96 120 96 120 96 120 96 120 96 120 96 120 MW (kD) 96 120 96 120 96 120 96 120 96 120 96 120 MW (kD)

48 72 48 72 48 72 48 72 48 72 48 72

Vorführender


Higher expression is observed in the The SDS-PAGE gel analysis only shows expression of the structural protein. Assembly of the structural protein is required to ensure VLP formation.

Processing & Assembly of Multiple Structural Proteins

VLP

http://www.virology.ws/2008/05/15/enterovirus-71-outbreak-in-asia/

Vorführender


What about multiple structural proteins

Multi-Protein VLP

• Enhanced expression and polyprotein processing and cleavage to structural proteins at lower temperature

Vorführender


We have seen enhanced expression….. The increase in yield of structural proteins correlated to increase yield of assembled VLPs.

Summary • No one baculovirus system consistently perform

better than the other • BEVS screen is critical to maximise protein and VLP

yield!

• Analysis of results from benchmarking exercise across different facilities ▫ Any particular trends observed?

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

Our track record Our expertise

Our capabilities Our platform

Thank You

Documents

BEVS Screen for Improved Protein Survival · Recombinant Protein Production Discovery, Translational, Preclinical Drug discovery, vaccinology, diagnostics, functional materials, targeting