AtaGenix
Technical Service

Insect Cell Expression System

Introduction

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


Vectors

Features

pFastBac1

Strong AcMNPV polyhedrin (PH) promoter for high level protein expression

Large multiple cloning site for simplified cloning

 

 

 

pFastBacHT

Strong polyhedrin (PH) promoter for high-level protein expression

 

N-terminal 6His tag for purification of recombinant fusion protein using metalchelating resin and aTEV protease cleavage site for removal of the 6His tag following protein purification

 

Vector supplied in 3 reading frames for simplified cloning

 

pFastBacDual

Two strong baculovirus promoters (PH and p10) to allow simultaneous expression of two proteins

 

Two large multiple cloning sites for simplified cloning

Common Expression Vectors and Features



Cells

Origins

Usages

 

Sf9

Sf9 cell line clone sub IPLBSF21-AE (Sf21), derived from the ovarian tissue of pupae of the fall fly worm (meadow nightshade)

 

Suitable for transfection, purification, production of high titer viruses, plate laying, and expression of recombinant proteins

 

Sf21

Sf21 cells are derived from the IPLBSF-21 cell line and also from the ovarian tissue of the pupa of the tsetse fly, Noctuella tetrasperma

Also perfect for transfection, purification, production of high titer viruses, plate laying and expression of recombinant proteins. Sf21 cells may express higher amounts of protein than Sf9 cells in some structures

 

Hi5

The Hi5 cell line was derived from plant studies at the Boyce Thompson Institute and from cabbage looper ovary cells and pink nightshade moth cells

Suitable for expression of recombinant viruses, often used for transfection and vacuolar purification, and also fit for expression of secreted recombinant proteins.

 

S2

S2 is a Drosophila late embryonic cell. Most of them are female tetraploid cells, but some are diploid cells

More suitable for recombinant protein  expression

Advantages

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.

Contents

Projects

Receivables

Contents

Time Frame

Deliverables

Insect Baculovirus Expression Protein

Gene Sequence

Vector

Gene Synthesis & Codon Optimization

Vector Construction

Virus Packaging & Amplification

Expression Purification Testing

Amplification & Purification

Reports

6-8 weeks

Protein Samples

Technical Service Report

Case Study

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. 3 P2 Expression Test of Protein A in Sf and Tn Cells - WB Validation (Reduced SDS-PAGE)
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.