recombinant protein production in baculovirus/insect ... · pfastbac dual two mcs in one vector...

Recombinant Protein Production in Baculovirus/Insect Expression System

Andy Liu, Ph.D.

[email protected]

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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


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


How to manipulate baculovirus/insect expression system?

How to improve protein production?

Case studies

Baculovirus/Insect Cell System Optimization


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


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




Case studies



Codon Optimization

Donor Plasmid Baculovirus Insect Cell Infection Construction

Amplification

Gene synthesis with codon optimization


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


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%




Case studies



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


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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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


GenScript’s Experience

GenScript has delivered over 5,000 proteins in four expression systems. Statistics showed

95% success rate for all protein projects.


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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Q&A

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Email: [email protected]

mailto:[email protected]

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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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recombinant protein production in baculovirus/insect ... · pfastbac dual two mcs in one vector...

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