Expression, Optimization and Production of Recombinant Proteins in Insect cells Using
Baculovirus
Francis Rajamohan
Protein and Cell SciencesPfizer Global Research and Development
Groton, CT
Outline● What is baculovirus● Why use baculovirus for protein expression● Baculovirus expression vector system (BEVS) ● Baculovirus expression host systems● Expression optimization● Post translational modifications● Scale-up of insect cells● High throughput expression● Summary
What is Baculovirus?1. Baculoviruses are enveloped, double- stranded
DNA (circular, supercoiled) viruses with rod-shaped nucleocapsids
2. Baculovirus life cycle involves two distinct forms of viruses, Budded Virus [BV ] and Occluded Virus [OV]
3. BV consists of a single nucleocapsid enveloped by GP64, a virus derived glycoprotein, and host membrane proteins
4. OV consists of multiple nucleocapsidsembedded in a protein matrix (polyhedrin matrix)
5. The most extensively studied baculovirus strain
is Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV).
6. AcMNPV only infects larval lepidopteransCourtesy: www.answers.com/topic/baculovirus
Baculovirus life cycle● Early Phase (0-6 h PI)● Virus enters cells by endocytosis
● Nucleocapsids migrate to nucleus
● Viral DNA is released
● Early gene expression starts
● Late Phase (6-24 h PI) ● Extensive DNA replication
● Progeny nucleocapsids leave nucleus
and acquire envelope as they leave
cytoplasm
● Production of budded virus
● Very Late Phase ● Decrease in the formation of budded virus
● Nucleocapsids acquire envelopes inside
nucleus to form MNPVs
● MNPVs are embedded in a matrix made
predominantly of the polyhedrin protein
and form occlusion bodies ● Courtesy: Dr. Linda Lua, The University of Queensland, Australia
This polyhedrin promoter is the major component of the baculovirus expression vector system
Why use Baculovirus for Protein Expression?
Higher level of gene expression (up to 50% of total cellular protein), in most cases, soluble and functionally active
Permits post-translational modifications
Phosphorylation
Disulphide bonds and proper folding
N-and O-linked glycosylation
Signal peptide cleavage
Easy to scale-up, insect cells are simple to maintain as suspension culture compared to mammalian cells
Inexpensive compared to other eukaryotic expression systems
Baculovirus - An important expression system in Structural Biology at Pfizer (Groton)
E. Coli 20 %
Insect cells 35 %
Mammalian 40%
Other 5 %
Baculovirus Expression Vector System (BEVS)
BEVS was pioneered by Dr. Max D. Summers, and Dr. Gale Smith in 1982
BEVS is based on replacement of a very late, non-essential, viral gene (polyhedrin), with a gene of interest
Most of the transfer vectors use either early (Ie1) or very late (p10, pPolyh) promoters
Modified and linearized AcMNPV DNA revolutionized the BEVS
For secreted proteins, HBM & gp67 are the most commonly used secretion signal
BEVS allows rapid cloning and expression of recombinant proteins in insect cells (Sf9, Sf21, Hi5)
300 bp
2.4 kb
Courtesy: Prof. Linda A. King, School of Biological and Molecular Sciences, Oxford Brooks Univ., Oxford, UK
Baculovirus expression vector system (BEVS)……Bac-to-Bac (Invitrogen™)
● E.coli lacZ gene inserted
at the polyhedrin locus
● Multiple Bsu361 restriction
sites introduced
● Bsu361 digestion results in
viral DNA incapable of
replication in insect cells
(∆ORF1629)
● Bsu361 digestion of DNA
is not 100%
● Plaque purification of
recombinant virus is
necessary
Courtesy: Prof. Linda A. King, School of Biological and Molecular Sciences, Oxford Brooks Univ., Oxford, UK
Baculovirus expression vector system (BEVS)……BacPAK6/BaculoGold (BD Biosciences/Clonetech)
Courtesy: Prof. Linda A. King, School of Biological and Molecular Sciences, Oxford Brooks Univ., Oxford, UK
Baculovirus expression vector system (BEVS)……BaculoDirect™ (Invitrogen™)
● Direct transfer of gene of interest by Gateway® technology
● Gene of interest is integrated into the polh locus of the viral DNA
● Integration mediated by integraseenzyme and specific attachmentsites (att)
● Selection of recombinant virusby ganciclovir (nucleoside analog)
● Phosphorylated ganciclovir, by HSV1tk, inhibits DNA replication of WTviral genome
● Requires multiple viral amplification● Restricted to the production of single
recombinant gene at a time
TK gene
Baculovirus expression vector system (BEVS)……flashBAC™/BacMagic (EMD/OET/Nextgen)
Courtesy: Prof. Linda A. King, School of Biological and Molecular Sciences, Oxford Brooks Univ., Oxford, UK
● Modified viral genome with a bacterial
artificial chromosome (BAC) at the polh
locus, allows the viral genome to be
maintained in E. coli
● A deletion in ORF1629 prevents replication
of non-recombinant, parental virus, in insect
cells
● Deletion of chiA gene has substantially
improved the efficacy of the secretory
pathway
● Amenable for automation
● No requirement for plaque purification
Comparison of Baculo Expression Systems
BacPAK6 / BaculoGold
Transfection
8-9 Days
BacPAK6 / BaculoGold
Plaqe assay
Sf9 cells8-9 Days
BAC-to-BAC
Transformation
Sf9 cells9 Days
DH10Bac
DNA pre. Transfection
BaculoDirect
Gateway LR
Sf9 cells5 Days
Transfection
flashBAC
Transfection
Sf9 cells5 Days
BAC-to-BAC
Transformation
Sf9 cells9 Days
DH10Bac
DNA pre. Transfection
BAC-to-BAC
Transformation
Sf9 cells 9 DaysDH10Bac
DNA pre. Transfection
BaculoDirect
Gateway LR
Sf9 cells5 Days
Transfection
BaculoDirect
Gateway LR
Sf9 cells 5 Days
Transfection
flashBAC
Transfection
Sf9 cells5 Days
flashBAC
Transfection
Sf9 cells 5 Days
Bac to Bac® Transfection
Remove DNA & add 25 mlfresh SF-900 II SFM mediumIncubate @ 27°C for 72h
Remove the medium (P0 virus)and Filter sterilize
100 µg Bacmid DNA +
100 µl CELLFECTIN in 4 mlSF 900 II medium
20 ml Sf-9 cells (1X106 vc/ml)SF-900 II SFM medium inT172 Flask & incubate for 45 minTo attach the cells
Add 10 ml of P0 virus to 1L sf-9 cells (2 X 106 vc/ml)Incubate for 72h
● BIIC Stock (1X107 vc/ml)
● Protein Purification
● Protein Characterization
● Activity Assay
● Crystal trial (if the
expression is >10 mg/L)
In 6 Days
Incubate @ 27°Cfor 5h
Transfection
Bac to Bac Transfection in Suspension Cells
Mix 100 µg of rBacmidDNA & 100 µl
CELLFECTIN in 4 ml SF900 II50 ml Sf9 cells (2.0 x 106 vc/ml)
■ BIIC Stocks
■ P0 Virus Stock
■ Expression analysis
■ Small scale purification
■ Activity assays
Cedex Cell analyzer
● Viability (>70%)
● Diameter (> 3 µ m
bigger)45 min @ RT
3 - 4 days
3 - 4 days
Baculovirus Expression Host Systems
TriEX™Intracellular protein productionSf-9 derivativeTri-Ex
EX-CELL 405Express Five SFMHyQ SFX
Secretion of recombinant proteinTrichoplusia ni(Ovarian cells)Hi-5
Sf-900 II/III SFMESF- 921HyQ SFX
Intracellular protein productionSecretion of recombinant protein
Spodoptera frugiperda(Pupal ovarian tissue)Sf-21
Sf-900 II/III SFMHyQ SFX
Recombinant baculovirus productionIntracellular protein expression Plaque assay
Spodoptera frugiperda(Pupal ovarian tissue)Sf-9
Growth MediumUseOriginCell line
Commonly used insect cells for protein production
Expression OptimizationTemperature and growth
Standard and Reduced TemperatureGrowth of Sf9 Cells
0 1 2 3 4 5 6 7
Sf9 - 20CSf9 - 24C
Day
Viab
le C
ell N
umbe
r
Viability of Sf9 Cells -Standard and Reduced Temps
0 1 2 3 4 5 6 785.0
87.5
90.0
92.5
95.0
97.5
100.0
Sf9 - 20CSf9 - 24CSf9 - 27C
Day
Viab
ility
(%)
Standard and Reduced TemperatureGrowth of Sf21 Cells
0 1 2 3 4 5 6 7
Sf21 - 20CSf21 - 24CSf21 - 27C
Day
Viab
le C
ell N
umbe
r
Viability of Sf21 Cells -Standard and Reduced Temps
0 1 2 3 4 5 6 785.0
87.5
90.0
92.5
95.0
97.5
100.0
Sf21 - 20CSf21 -24CSf21 - 27C
Day
Viab
ility
(%)
2.0×10
4.0×10
6.0×10
8.0×10
1.0×10
1.2×107
7
6
6
6
6
2.0×10
4.0×10
6.0×10
8.0×10
1.0×10
1.2×107
7
6
6
6
6
Sf9 - 27C
M 17 45 67 93 117 17 45 67 93 117Hours Post Infection
191
97
64
51
39
28
20°C 27°C
137 kDa
Protein expressed in Sf-21 cells
Expression OptimizationTemperature and Expression/Stability
● Expression levels may vary depending on target protein
(-)
Na
Bu
(-)
Na
Bu
+2
mM
Na
Bu
24h
pi
+2
mM
Na
Bu
48h
pi( -
)N
aB
u
(-)
Na
Bu
+2
mM
Na
Bu
24h
pi
+2
mM
Na
Bu
48h
pi
64
51
39
Protein expressed in Sf-9 cells
27°C 24°C
0
20
40
60
80
100
120
0 24 48 69
sf9/Sf-900II/MOI 1sf9/Sf-900II/MOI 5
Hi5/Express 5/MOI 1Hi5/Express 5/MOI 5Hi5/Excell 405/MOI 1
Hi5/Excell 405/MOI 5R
elat
ive
A ct iv
ity
Time Post Infection (hr)
Expression OptimizationMOI and Media
● MOI between 1& 5 did not have significant effect● Growth media may affect expression levels● Expression levels may vary depending on target protein
Expression OptimizationSecretion Hosts
30
50
75100
kDa
Sf9 cells Hi5 cells
No Difference in Expression
31-S
f21-
Med
Marke
r
Cont
. Med
ia31
-Hi5
Med.
39-H
i5 Med
.
97
64
51
39
28
Sf21 is better than Hi5
● Expression levels may vary depending on target protein
Commonly used fusion tags in insect cells
1. Mild elution conditions2. Works under both native and denatured condition3. Inexpensive affinity resin
IMAC6 kDaSplit SUMO(6X-His)
1. Variable efficiency of enzymatic biotinylation2. Mild elution conditions3. Expensive affinity resin
Strep•TactinStreptavidin
8 aaStrep
1. High metabolic burden2. High specificity3. Inexpensive affinity resin4. Mild elution conditions / high binding capacity
Glutathione affinity26 kDaGST
1. Low metabolic burden2. High specificity3. Expensive affinity resin4. Harsh elution conditions / low binding capacity
Anti FLAG M2 AB8 aa(DYKDDDDK)FLAG
1. Most common purification tag2. Low metabolic burden3. Mild elution conditions4. Works under both native and denaturing conditions5. Inexpensive affinity resin / high binding capacity
IMAC6-10 aaHis
CommentsColumnSizeTag
Expression Optimization
● The tags may or may not effect expression level and solubility
Expression OptimizationExamples of Tag-Purification
● Expression levels may vary depending on target protein
Sf-9 Sf-21
FLAG-Tagged
191
97
64
51
39
GST-Tagged
Sf-915010075
50
37
250
25
Fusion
Target
Thrombin
GST
191
His-Tagged
Sf-9
97
64
51
39
28
Fc-Fusion
Hi-5200
97
66
55
37
116
31
Column: Anti FLAG M2 ABElution: Anti FLAG peptide
Column: GSTrap®Elution: Red. glutathione
Column: HiTrap® ProteinAElution: 0.1M Citric acid
(pH 3.5)
Column: HisTrap®Elution: Imidazole
Expression OptimizationExpression & purification of BAP-Tagged protein from Sf-9 cells
6.33e4 cpsBioSpec Reconstruct for +Q1: 6.43 min (7 scans) from Griffor 4/19/02 003, subtracted (scans 46 to 51)
28377.028695.0
28944.029819.0 30297.0
31002.0
32187.032682.0
28500 29000 29500 30000 30500 31000 31500 32000 32500
Mass, amu
10000
20000
30000
40000
50000
60000
Inte
nsity
, cps
Theoretical mass withOne Biotin (226): 30996
pMCG2336243bp
birA
BAP tag
GmR
AmpR
f1 intergenic region
SV40 polyA
HSV tk polyA
p10pPolh
Tn7R
MCS
3136
21
5566
T S AD FT 1 2 3 4 5
Column: SoftLink™ Avidin ResinElution: Avidin
Courtesy: Avidity.com
Post Translational Modifications
● Most studies suggest that insect cell-derived recombinant glycoproteins fail to acquire peripheral sugars, such as sialic acids
● Sialic acids play a key role in many cell-cell interactions, immunological reactions and in the clearance of circulating glycoproteins
● Dr. Donald L. Jarvis has engineered several insect cells to produce recombinant proteins with terminally sialylated N-glycans just like mammalian system.
Post Translational Modifications
E.coli
50
35
25
15
29
Pichia cells
50
35
25
15
2931
Insect cells
50
35
25
15
2930
Sample from insect cells
50
35
25
15
CarbohydratePositive
CarbohydrateNegative
Western Blot for Glycan Detection
● No Glycosylation by E. coli● Significant glycosylation by Yeast and Insect cells
Glycosylation:
Coomassie SDS-PAGE: Anti-FLAG M2 affinity purified samples
Post Translational Modifications
Removal of glycosylation site by mutation
● Removal of glycosylation site(s) might reduce expression levels
WT WTGly
cosy
latio
nM
utan
t
Gly
cosy
latio
nM
utan
t
50
75100
30
15
Phosphorylation:
Post Translational Modifications
● High-level expression of protein kinases leads to hyperphosphorylation and hetrogeniety of the recombinant fusion protein
● Phosphorylation of serine residues adjacent to His-tags (MHHHHHHSSGLVPRGS) of several commercial vectors have been observed
● Hyperphosphorylation of the serine residues in the tag leads to aggregation and resistance to thrombin cleavage
● Treatment with alkaline phosphatase partly restore sensitivity to thrombin● Treatment of insect cells with okadaic acid (protein phosphatase inhibitor)
leads to extensive phosphorylation at the tag sequence
Wave Bioreactor
No mixerNo sterilizationNo cleaningNo pipingMinimal downtimeNo maintenanceFrom 1 to 100 liters
Conventional Method
Scale-up of Insect Cells
Scale-up of Insect Cells
1-BIIC Vial/10-L
Two 1L Sf9 cells grownto 1.0 x 107 vc/ml
Mixed in 100 mlSF 900 IISFM
● Starting cell density 2.0 x 106 vc/ml● Temperature 27°C● Rocking 25 rpm● Air 0.1 – 0.2 lpm● Harvest after 72 h or when the viability
is between 80 to 90%
Wave system 20/50 EH
High Throughput Expression
● Sf9 cells (static culture)
● Routinely seed wells 2 x 105 per well (0.4 ml Sf900II) ● Cells dispensed using the baculoworkstation
● Co-transfection (polystyrene)
● polystyrene tubes / plates● 100 ng flashflashBACBAC DNA● 500 ng transfer vector● 2µg Lipofectin or Cellfectin
● Incubation
● 28Cْ / 5 days
Nextgen Baculo workstation
Courtesy: Nextgen
Baculoworkstation Pros and ConsPros:• Provides significant walk away time on a daily basis
• Increases throughput
• Reproducibility
• Reduces the risk of human error
• High accuracy dispensing systems
Cons:• Fairly expensive
• Not amenable for scale-up (small pipetting volumes)
• Not usable for suspension cultures
• Only replaces some of the manual steps
Summary● Advances in commercially-available BEVS permits simple
recombinant virus production and expression
● Most insect cell lines have been adapted to serum-free media that support faster cell growth, higher virus titer and higher protein yield
● Easy and inexpensive to scale-up
● Amenable for automation
● The availability of transgenic insect cell lines expressing humanized protein glycosylation pathways offers a way to overcome the potential problem of native glycosylation of human proteins