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Recombinant Protein Production in Baculovirus/Insect Expression System
Andy Liu, Ph.D.
andy.liu@genscript.com
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Table of Contents
Expression system selection
Baculovirus/insect cell system optimization
Trouble shooting & case studies
GenScript protein services
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Bacteria E. Coli strains
1. Well established
2. Simple genetics
3. Easy scale up
4. Speed
5. Low cost
6. Equipment
Insect cell
Sf9, High5, Sf21
1. PTMs resemble mammalian system
2. Soluble proteins
3. Good secretion
4. Virus infection
Mammalian cell
CHO, HEK, COS
1. Comprehensive PTMs
2. Soluble proteins
3. Low expresser
4. Expensive
Yeast
S. cerevisieae，
P. pastoris
1. PTMs
2. Low cost
Protein Expression Systems - Pros
PTMs: post-translational modifications
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Bacteria
E. Coli strains
1. Lack of efficient PTMs
2. Inclusion body
3. Difficult to express higher MW proteins
Insect cell
Sf9, High5, Sf21
1. Long production time
2. Relative high costs
Mammalian cell
CHO, HEK, COS
1. Long production time
2. High media costs
Yeast
S. cerevisieae，
P. pastoris
1. Improper glycosylation
2. Excessive glycosylation
Protein Expression Systems - Cons
PTMs: post-translational modifications
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92%
3.8% 2.06% 1.03% 0.29% 0%
20%
40%
60%
80%
100%
E.coli Insect Yeast Mammalian Cell Free
PDB entries reflects dominance of Insect expression
Insect is the Top Choice in Eukaryotic Cells
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Number of entries in the PDB by expression system as a percentage of total number of chains with an identifiable expression system, as of April 15, 2014. All values are approximate. Reference - http://www.rcsb.org/pdb/home/home.do
The most efficient & cost-effective way
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How to manipulate baculovirus/insect expression system?
How to improve protein production?
Case studies
Baculovirus/Insect Cell System Optimization
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Baculovirus/Insect Expression System
Donor plasmid
~12-15 days
Construction Amplification
Baculovirus
~2-4 days
Infection
Insect cell
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Bac-to-Bac® Baculovirus Construction
Bac-to-Bac: from Bacterium to Baculovirus
powerful method for Baculovirus construction
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Transformation
Competent DH 10BacTM E.coli Cells Recombinant Donor Plasmid
Mini-prep Bacmid
Recombinant Bacmid DNA
Transfection
Adherent sf9 cells Baculovirus P1
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Stage Production Stock
Recombinant E.coli strain (glycerol stock) -80 oC, more than 1 year
Recombinant Bacmid DNA 4 oC, up to 2 weeks
Baculovirus P1 4 oC, 3-6 months
Bac-to-Bac® Baculovirus Stock
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Baculovirus Amplification
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1. Generally viral titer increases from 106 to 108 pfu/ml by after two generations.
2. The infected sf9 cells and supernatant (for secreted protein) will be used to evaluate the target protein expression timely.
3. Baculovirus could be amplified easily for scale-up production.
P1
Amplification
sf9 cells
P2
Amplification
sf9 cells
P3
Expression evaluation - +
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Protein Production with Baculovirus/Insect Cells
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10-100 L (Wave)
Cell pellet
Supernatant
40 ml-10 L
(Flask) Infection
Baculovirus
Temperature: 27 oC
pH: 6.1 to 6.4
Viability: >95%
CO2: No
O2: Yes
Cell density: 1*106 to 2*106
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How to manipulate baculovirus/insect expression system?
How to improve protein production?
Case studies
Baculovirus/Insect Cell System Optimization
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Codon Optimization
Donor Plasmid Baculovirus Insect Cell Infection Construction
Amplification
Gene synthesis with codon optimization
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Vector Note
pFastBacTM 1 No extra residue, easily edit for optional tag fusion
pFastBacTM HT Cleavable N-terminal 6 X His tag
pFastBacTM Dual Two MCS in one vector
pDEST20 Cleavable N-terminal GST tag
Donor Plasmid
Bac-to-Bac® Baculovirus Expression System
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Expression Strategy
Gp67: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA
HBM: MKFLVNVALVFMVVYISYIYA
Gp64: MVSAIVLYVLLAAAAHSAFA
Secreted Expression Intracellular Expression
—His tag —TEV—your gene—
Kinds of tags are available: His, GST, Fc, Flag, …..
—signal peptide—His tag —TEV—your gene—
Multi-subunit complex
pFastBac Dual Two MCS in one vector co-expression by multi Baculovirus
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Optimization for Expression
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Cell line
Case by case, protein yield is variable in these cells.
Cell line Doubling time Virus Amplification Protein expression Sf21 24-30 h Yes Yes
Sf9 24-30 h Yes Yes
High FiveTM 18-24 h No Yes
MimicTM Sf9 24-30 h No Yes
Time course post-infection
Secreted proteins:30 to 72 h. Non-secreted proteins: 48 to 96 h.
Multiple Of Infection
MOI: 1 to 5 Fresh virus: no more than 3 weeks
0.1% to 0.5% FBS or BSA
Culture condition
Viability: >95%
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How to manipulate baculovirus/insect expression system?
How to improve protein production?
Case studies
Baculovirus/Insect Cell System Optimization
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Case Study
Three ~100 KDa subunits were co-expressed in High 5 cells
Alexander Pflug, 2014, Nature
Intracellular expression
Influenza virus polymerase Huntingtin protein
~350 KDa was expressed in sf9 cells
Intracellular expression
Insect cell Ihn Sik Seong, 2010, HMGs
Mammalian cell Bin Huang, 2015 Plos one
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Case Study
Sobhan Roy, 2014, PNAS
TCR (α chain + β chain)
2 subunits were expressed in High 5 cells
Secreted expression
Glycosylated site
Kinase, Cytokines, membrane protein ……
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Table of Contents
Expression system selection
Baculovirus/insect cell system optimization
Trouble shooting & case studies
GenScript protein services
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Make Research Easy 21
Package Name Cat. No. Specifications Price Comments
PROtential Package SC1653-I One subcloning strategy, only do transfection
$400 Deliver expression evaluation report
Customized Package SC1810 Can start from gene synthesis, subcloning, virus generation or amplification
Quote Charge for the performed service
InsectPower™ Guaranteed Package
SC1640 Two subcloning strategies, 1 mg, 75%-85% purity protein
$3950 Absolutely free if we cannot deliver protein.
FragPower™ Guaranteed Package
SC1686 Co-expression light and heavy chain
$3399 Deliver 3 mg, >90% purity Fab
BV Stock Package
SC1261
One subcloning strategy, Baculovirus preparation and expression evaluation
$1900 Deliver 10 ml, >10^7 pfu/ml virus
SC1835 $2250 Deliver 100 ml, >10^7 pfu/ml virus
SC1840 $2700 Deliver 100 ml, >10^8 pfu/ml virus
Services for Baculovirus/Insect Expression System
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GenScript’s Experience
GenScript has delivered over 5,000 proteins in four expression systems. Statistics showed
95% success rate for all protein projects.
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Protein Services at GenScript
Mammalian Expression
Transient Expression, Stable Cell Line Development
Process Development,
GLP-Compliant Bioprocess
Bacterial Expression
BacPower™ Guaranteed, BacPower™ Customized,
Fast Gene-to-Protein,
FragPower™ Antibody Fragments, Fermentation
Recombinant Antibody Services
MamPower™ Guaranteed Antibody, FragPower™
Antibody Fragments,
Guaranteed Gram Level Ab Cell Line,
High Throughput Antibody Purification,
Antibody Service Selection Guide
Insect Expression
InsectPower™ Guaranteed Protein, BacuVance™
Baculovirus Expression,
GLP-level Insect Protein Expression,
FragPower™ Antibody Fragments
Other Protein Services
Yeast Expression, HTP Protein Variants, Chemical
Protein Synthesis, Endotoxin Removal, Custom Protein
Purification,
Protein Characterization, Protein Refolding
Resources and Technical References
ProtBank™ Protein Database, WoLF PSORT II,
Protein Learning Resources, Case Studies,
Downloads, FAQs, Protein News Page,
Publications, Technical Guide, Technical Notes
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Relevant Services at GenScript
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GenScript
Products & Services
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Gene
Peptide
Protein
Antibody
Discovery Biology
Cell Line
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Thank you for your participation
We wish you all success in your Research
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To view other webinars in the GenScript Webinar Series @
http://www.genscript.com/webinars.html
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