expression, optimization and production of recombinant … · expression, optimization and...

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


Baculovirus expression vector system (BEVS)……BacPAK6/BaculoGold (BD Biosciences/Clonetech)


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)


● 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


BAC-to-BAC

Transformation

Sf9 cells 9 DaysDH10Bac


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


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


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.


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


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:


● 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

Acknowledgements● Alison Varghese● Dr. Colin Robinson● David Wasilko● Dr. Ing-Kae Wang● Jim Duerr● Kim Fennell● Matt Griffor● Dr. Peter LeMotte● Pat Loulakis● Timothy Subashi

expression, optimization and production of recombinant … · expression, optimization and...

Documents