Insect baculovirus expression system (BEVS) is an important eukaryotic expression system that utilizes baculoviruses as vectors for exogenous genes and expresses the exogenous proteins by infecting insect cells or insect larvae.

1. Advantages

a. Good safety

b. Large capacity to carry exogenous genes

c. High expression level of exogenous target proteins

d. The expressed eukaryotic gene products can be correctly folded and modified, and thus have good biological activity.

2. Advantages of Insect Expression Systems Over Mammalian Cells:

a. Relatively simple culture conditions, no need for CO2

b. Not easily contaminated by bacteria

c. Capable of post-translational glycosylation modification of proteins

d. Simple conditions for mass production, easier to obtain large amounts of protein

Common Expression Vectors and Features

1.AtaGenix has created a fast, simple and efficient baculovirus expression platform by adopting classic Bac-to-Bac technology; The technology can produce recombinant viruses quickly and without the need for null spot analysis, greatly improving efficiency.

2. Short cycle time and high throughput for protein production.The process of cells from small-scale culture to large-scale fermentation can be realized, so that the protein product can even reach the level of 100mg/L.

3. Provide free virus particle amplification and storage service, the storage period is up to 2 years.

Case 1 Insect Barcovirus System Expressing Large Protein A (272 KD)

AtaGenix expressed protein A of 272 KD by using insect-rodovirus expression system with C-terminal fusion of 6*His tag. After the P1 expression test, Sf cells and Tn cells were transfected to determine MOI, the optimal expression conditions were selected for P2 expression test and 200 ml expression purification, and the final protein A purity was 90%, and the yield was 5mg/L. The final protein A was obtained from AtaGenix, and the final protein A was obtained from Sf cells.

Fig. 1 Protein A P1 Expression Purification Test Results ( Reduced SDS-PAGE )

A.Coomassie Blue Staining  B. WB (WB (ECL Luminescence, Anti-His Labeling Antibody).

ø. Negative Control Medium. 1 and 2. Trzansfected Clones 1 and 2.

Fig. 2. P2 Expression Test of Protein A in Sf and Tn Cells (Reduced SDS-PAGE).

Coomassie Blue Staining  Ø. Negative Control. D2-D3. Days after Transfection. +. Positive Control.

Fig. 4 Protein A Purification QC (Reduced SDS-PAGE)

A.Coomassie Blue Staining      B. WB (ECL Luminescence; Anti-His Tag Antibody). MW. Molecular Weight Labeling

Case 2 Insect-barcovirus System Expression G Protein-coupled Receptor (Protein B)  

AtaGenix used an insect-baculovirus expression system, and the N-terminal fusion of 10*His and Flag tags expresses G protein-coupled receptor-protein B (7 transmembrane regions, 2 glycosylation sites) with a size of 42 KD. After the P1 expression test, the virus was amplified and transfected into Sf cells and Tn cells to determine the MOI, the optimal expression conditions were selected for P2 expression test and 200 ml expression purification, the final protein B was determined with biacore, and the affinity was at uM level.

Figure 1.Protein B P2 Expression Purification Test (Sf and Tn cells)

Reduced SDS-PAGE and Coomassie Blue Staining

Ø.Negative Control.D2-D3.Days after Transfection

Medium: Culture Medium.NPE.Soluble Supernatant.DPE.Precipitation

Fig. 2 WB Validation of the P2 Representative Reach Test (Sf and Tn Cells; ECL Luminescence; Anti-His Labeling Antibody).

Ø.Negative Control.D2-D3.Days after Transfection

Medium: Culture Medium.NPE.Soluble Supernatant.DPE.Precipitation

Fig. 3 Protein Purification QC (Reduced SDS-PAGE;Coomassie Blue Staining)

 MW. Molecular Weight Labeling

Figure 4. Protein B and Ligand Affinity Assay Results