Insect cell protein expression

Protein expression form

    Get a high yield protein expression in a eukaryotic system at affordable price! Trust our experts to optimize all the parameters and skyrocket the yield of your insect cell expression!

    Why choose ProteoGenix for insect
    cell protein expression?

    Insect cell expression optimization
    Access a complete toolbox

    We propose a wide variety of cell lines and expression vectors to optimize your insect cell protein expression.

    protein expression expertise
    Unrivalled protein expression expertise

    We routinely perform protein expression since 2003. 1500+ proteins expressed so far.

    baculovirus production protocol
    Optimized protocols

    Our baculovirus protein expression service includes optimization of several parameters such as choice of insect cell line, multiplicity of infection and incubation time.

    gene to baculovirus expression
    Integrated solutions

    Choose a unique partner from gene synthesis to protein expression.

    gene for insect cell expression
    Codon optimization

    Benefit from ProteoGenix’s gene synthesis expertise to get an optimized gene for insect cell expression.

    baculovirus expression expertise
    PhD account managers

    At ProteoGenix, you benefit immediately from the expertise of a PhD account manager!

    Our insect cell protein expression process

    optimized gene synthesis for insect cell expression
    • Expression vector construction (optional)

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

    baculovirus production
    • 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 and purification
    • Protein expression scale up and purification

    Protein expression and purification with optimized conditions previously determined.

    Our insect cell expression workflow

    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


    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:


    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.



    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.


    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



    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.