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Our insect cell protein expression process

Expression vector construction (optional)

  • Gene design including codon optimization
  • Gene Synthesis
  • Subcloning in an expression vector

Virus production and amplification

  • Transformation of the plasmid in competent E. coli cells to generate the Bacmid DNA
  • Transfection of insect cells with the recombinant Bacmid
  • Generation of the P1 Baculovirus stock
  • Baculovirus amplification(P2 stock)
  • Determination of virus titer

Small scale protein expression

  • Transfection and selection of yeast clones
  • Determination of top expressing clones
  • Expression Optimization
  • SDS/page + WB if necessary
  • Purification tests

Protein expression scale up and purification

  • Protein expression and purification with optimized conditions previously determined.
Step Content Timeline Deliverables
Virus production and amplification
  • Transformation of the plasmid in competent E. coli cells
  • Transfection of insect cells with the recombinant Bacmid
  • Generation of the P1 Baculovirus stock
3 to 4 weeks
  • Intermediate report
Protein expression scale up
  • Baculovirus amplification (P2 stock)
  • Infection of insect cells with P2 Baculovirus stock (for protein expression evaluation)
  • Optimization of several expression conditions (MOI, incubation time…)
  • SDS/Page + WB if necessary
  • Purification
2 weeks
  • Test protein sample
Virus production and amplification
  • 1L protein expression and purification
3 to 4 weeks
  • Final detailed report
  • Purified protein
  • Gene synthesis including codon optimization for protein expression and subcloning in an expression vector
  • Size exclusion chromatography
  • Polishing (DNA, HCP, endotoxins)
  • Endotoxin removal
  • Analytics : WB, analytical SEC

Case study:

Example of insect cells production of a Beta-Glucosidase

Service Performed

We were asked by a customer to produce a recombinant Beta-Glucosidase protein using a baculovirus expression system (Insect cell production). Our customer ordered a full Insect cell package, including small-scale expression and purification tests followed by a 1L pilot production and purification. We reached a yield of 370 mg/L and delivered 60 mg after small-scale expression. 1L pilot production was no longer useful and was therefore not charged to our customer. The services carried out included:

Molecular Biology

The cDNA coding for Beta-Glucosidase protein was chemically synthesized after sequence optimization for insect cells expression. It was then subcloned in a proprietary expression vector. A sequence coding for a 6His tag was added for further purification.

Expression Tests

P1 Virus Stock Generation

Figure 1. Analysis of insect cell expression during P1 generation.
Left. Coomassie Blue Staining. Right. WB with anti-His antibody (ECL substrate) “-“. Un-transfected
control culture. 1 and 2 are transfections with 2 different Bacmid clones

Conclusion of P1 virus generation step

A protein band matching with Beta-Glucosidase protein molecular weight is observed by SDS-PAGE in native protein extracts. This result is confirmed by Western blot (Figure 1, green arrows). Recombinant bacmid clone No1 was used for expression tests.

Virus Amplification And Expression Tests

Figure 2. Protein Expression Tests with P2 stock.
Coomassie Blue Staining. Left. Cell type 1.Right. Cell type 2. “-“. Negative control culture.

Conclusion of expression tests

Expression tests with P2 confirm that the target is present at high level in native protein extracts (Figure 2, green arrows). Optimal conditions for native expression: Figure 2, red arrow = loi

Purification Tests


Figure 3. Protein small-scale purification test. Coomassie Blue Staining.
Left. Purification profile. Right. Final QC of purification pool
IN. Input. FT. Flow through. W1-W3. Washing steps. E1-E9. Eluted fractions.

Conclusion of small-scale purification tests

Recombinant Beta-Glucosidase protein can be produced and purified in native conditions. The purity is ≥ 95%, and the production/purification yield is approximately 370mg/L. Baculovirus expression system was a perfect choice for this protein.

What are the advantages of insect
cell protein expression?

Baculovirus expression systems are eukaryotic expression systems. This confers to these systems all the advantages conferred by their eukaryotic nature such as:

  • Proper protein folding: insect cells integrate the whole protein folding machinery characteristic of eukaryotic expression systems. Baculovirus is the most commonly used expression system for structural analysis (e.g. determination of 3D protein structure).
  • Post-translational modifications: baculovirus expression systems are able to make post-translational modifications such as glycosylations and phosphorylations. These modifications confer improved solubility properties to the protein expressed compared to E.coli protein production and are very close to those found in mammalian cells.
  • Ability to express large protein complexes of mammalian proteins.

They even include unique advantages compared to other eukaryotic systems such as:

To conclude, insect cells expression represents the best option for researchers and companies willing to produce their protein in a eukaryotic expression system (with glycosylation profiles close to those obtained in mammalian cells) at affordable price.

Optimizing baculovirus
protein expression

There are many factors influencing the yield of baculovirus protein expression. One of the most important is the multiplicity of infection (MOI). Multiplicity of infection refers to the number of virus to add to cells.
There are mainly two processes used for baculovirus protein expression:

  • Low MOI process: this process refers to a two-steps baculovirus infection process. In the first phase, cells are inoculated with a MOI<1 resulting in only a partial infection of the cell culture. This means that uninfected cells continue to proliferate and that infected cells produce the recombinant protein and new virus particles. In this case, a second infection is necessary resulting in a high density infected cell culture. This method leads to longer processes but usually increases the yield of protein expression as more cells are available to produce the recombinant protein.
  • High MOI process: the high MOI baculovirus infection corresponds to a one-step process where an excess amount of virus (MOI>1) is added to the cell culture.

The yield of insect cell protein expression can also be enhanced by optimizing the incubation time. The goal of this step is to determine at which incubation time the cell culture should be harvested. Basically, cell culture should be harvested before protein degradation starts.

As each project represents a unique challenge, ProteoGenix tests several cell lines, multiplicity of infections, incubation times and culture media (under request). Contact our PhD account manager to get your custom offer.

Baculovirus expression
system principle

The baculovirus expression system is based on the infection of insect cells with baculovirus. Baculovirus are insect pathogens controlling the insect population in nature. They present a biphasic replication cycle driven by two forms of the virus:

  • Budded virus: the form necessary for the infection of insect cell culture.
  • Occlusion derived virus composed of a large amount of P10 and polyhedrin.

This latest form of the virus is not necessary for infection of insect cell cultures. Thus, the polh and p10 promoters can be exploited for recombinant protein expression. This is the basis of the baculovirus expression system.