Insect Protein Expression

Infection of high-density insect cell cultures with baculovirus expressing target genes is one of the most versatile and powerful systems for eukaryotic expression of recombinant proteins. The insect expression system maintains the majority of eukaryotic post-translational modifications and protein folding. It is often the preferred approach for producing kinases and steroid receptors. These proteins may remain inactive when produced in bacterial cells due to lacking proper phosphorylation or misfolding, respectively.

Highlights

Maintains essential eukaryotic protein modifications
Multiple insect cell lines are available
Useful for producing large protein targets
Suitable to produce secreted, intracellular, and membrane-bound proteins

Service Details

Insect cell protein expression workflow

Price and turnaround Price Match

Service Module	Brief Description	Deliverables	Price (USD)	Turnaround
Vector design and cloning	Vector design includes fusion partner selection, purification tag selection, and codon optimization. This is followed by gene synthesis and cloning into the baculovirus transfer vector, such as the pBV.	E. coli glycerol stock	From $189	1-2 weeks
Pilot-scale expression and purification	Baculovirus will be generated and amplified.	0.1-0.5 mg purified proteins (if feasible) COA	From $2,400	7-8 weeks
Pilot-scale expression and purification	Insect cells, such as Hi-5 or Sf9 cells, will be infected with the baculovirus. If feasible, 0.1-0.5 mg of purified target proteins * can be produced, and quality control (QC) will be performed.	0.1-0.5 mg purified proteins (if feasible) COA	From $2,400	7-8 weeks
Production-scale expression and purification	Scaled up production of >100 mg of purified proteins.	Up to 100+ mg purified proteins COA	Please inquiry

* Purification methods will be determined based on the construct design and protein characteristics.

Shipping and storage

The default deliverable for vector cloning is E. coli glycerol stock. Upon request, small amounts of plasmid DNA can be provided if available without additional charge. Most of our recombinant proteins are delivered as frozen liquid on dry ice. Upon receipt, they should be stored at -20°C to -80°C under sterile conditions. Recombinant proteins typically remain stable for up to a year if stored properly. Additionally, we recommend aliquoting the recombinant proteins upon receiving and avoiding freeze-thaw cycles.

Quality control (QC)

All vectors cloned by VectorBuilder come with a 100% sequence guarantee. All recombinant proteins produced by VectorBuilder undergo stringent quality control. Default QC for most systems includes 1) validation of the protein expression vector by restriction digestion analysis and Sanger sequencing; and 2) determination of protein concentration and purity by A260/280 measurement and SDS-PAGE. Common additional QC services are shown in the table below, which can be provided upon request.

Additional QC Services	Method
Endotoxin test	LAL
Protein characterization	Western Blot
	Intact MS (reduced)
	SEC-HPLC
	SEC-MALS
	Protein N-terminal sequencing
	Host cell protein test
Tag removal	Protease digestion
Kinetics and affinity analysis	Octet
Kinetics and affinity analysis	Biacore
Pathogen testing panel for rodents

Technical Information

Vector system

To express the recombinant protein in the insect system, the gene of interest (GOI) is first cloned into VectorBuilder’s pBV vector under the control of a strong promoter. This entire expression cassette is flanked by the Tn7 transposon terminal elements, Tn7L and Tn7R. This vector is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase. The transposase would then mediate the transposition of the region flanked by Tn7R and Tn7L on the pBV vector, which contains the expression cassette for the gene of interest and a drug-resistant gene, into the attTn7 docking site of the bacmid. Colonies containing recombinant bacmids can be identified by drug selection and blue/white screening (non-recombinant colonies are blue due to lacZ expression whereas recombinant colonies are white due to disruption of lacZ by transposon insertion). Purified bacmid DNA can then be used to transfect insect cells to generate live baculovirus (P1). The P1 baculovirus is then amplified in insect cells to achieve a suitable titer, which can be used to infect insect cells to produce the recombinant protein of interest.

For more information regarding VectorBuilder’s baculovirus protein expression vector system, please visit our vector guides.

Figure 1. Map of a baculovirus-insect recombinant protein expression vector.

Case study

Figure 2. Characterization of a recombinant protein produced in insect cells. (A) SDS-PAGE analysis shows the molecular mass of the recombinant protein, and the purity was determined to be ≥ 95%. (B) The purity was determined to be ≥ 95% by SEC-HPLC. (C) The biological activity of the recombinant protein was measured by a cell proliferation assay. The ED₅₀ is between 2 and 11 ng/ml.

How to Order

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Customer-supplied vectors

If customer-supplied materials are needed, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder, which may incur $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.

Resources

FAQ

Which recombinant protein expression system should I choose?

All recombinant protein expression systems have their pros and cons which should be taken into consideration while selecting the optimal system for your project. The table below summarizes the pros and cons of each system.

Recombinant Protein Expression System	Pros	Cons
Bacteria	Cost-effective Short production time Technically simple Can be scaled up easily High protein yield	Proteins lack post-translational modifications Codon usage is different from that of eukaryotes Difficult to express certain proteins due to the accumulation of inclusion bodies Some proteins are toxic and can inhibit bacterial growth
Mammalian cells	Produces proteins in their most natural state with all necessary post-translational modifications and proper folding Suitable for secretory and membrane proteins Most appropriate for producing therapeutic proteins	Long production time Requires complex growth conditions Scaling up can be difficult Not suitable for intracellular proteins due to low yield
Insect	Performs majority of complex post-translational modifications and protein folding Suitable for secretory, intracellular, and membrane proteins Can be used for producing large protein complexes High levels of protein expression compared to other eukaryotic expression systems Highly scalable due to high-density, suspension cell cultures	Long production time Requires complex growth conditions Technically challenging
Cell-free system	Time-efficient synthesis completed in 3 hours No need for live cells in the production of toxic, complex, or unstable proteins Suitable for high-throughput protein expression and screening Easy procedure compatible with automated processes Easy to optimize the conditions	Limited modifications are available

Which protein tag should I use?

Tags are frequently utilized for recombinant protein production. They streamline the purification process, and certain tags have demonstrated improvement in protein solubility, yield, or purity. If the tag is attached to the protease cleavage site, it can be removed after purification, and the efficiency of cleavage varies depending on the target protein. The careful selection of an appropriate tag is crucial for downstream protein expression and purification. The table below provides an overview of commonly used tags along with their advantages and limitations.

Tag	Commonly Applied System	Advantages	Limitations
GST	Bacteria, insect	Largely preserve the native structure of the protein Enables protein purification under mild conditions Easy cleavage Enhances the solubility and expression of the protein	Large tag size Dimerization may impact the target protein Not suitable for purifying proteins under denaturing conditions
His	All	Small tag size, therefore less impact on the protein Low cost for metal-affinity chromatography Low immunogenicity Suitable for purifying proteins under denaturing conditions	Other endogenous metal-binding proteins in microbial hosts may be co-purified, therefore optimization is usually required Does not facilitate protein folding and solubility
SUMO	Bacteria, insect	Facilitates protein folding Enhances the solubility and expression of the protein	May undergo non-specifically cleavage by other bacterial proteins Not suitable for purifying proteins under denaturing conditions
Flag	Mammalian cells, insect	Small tag size, therefore less impact on the protein Easy detection Low non-specific binding	Poor efficacy for purification
MBP	Bacteria, insect	Enhances the solubility and expression of the protein	Large tag size Not suitable for purifying proteins under denaturing conditions
Fc	Mammalian cells, insect	Enhances the solubility and expression of proteins Suitable for secreted protein	Large tag size

For more information regarding tags, please visit protein tags.

Which insect cell line should I use for the expression of my protein?

The Sf9 and Sf21 cells, derived from the fall armyworm Spodoptera frugiperda, and the Hi-5 cells, derived from the cabbage looper Trichoplusia ni, are the most widely used insect cell lines for baculovirus generation and recombinant protein expression. The table below summarizes the characteristics of each cell line.

Hi-5	SF9, SF21
Achieves high-quality baculovirus production Optimal for the production of secreted recombinant proteins Compatible with suspension cell culture Tends to develop irregular monolayers during growth in adherent cultures, leading to challenges in plaque identification	Achieves high-quality baculovirus production Suitable for all types of recombinant protein production Compatible with both monolayer and suspension cell cultures

Related Services

Vector cloning
Plasmid DNA preparation
Baculovirus packaging