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Baculovirus-Insect Cell Expression System (BICES) has been widely used for the production of recombinant proteins. It is one of the most powerful, robust, and versatile eukaryotic expression systems available. the BICES offers multiple advantages for protein production in a variety of applications, such as:

• expression of large cDNA fragment;
• expression of cell signaling proteins, cytokines,  kinase and membrane proteins; 
• virus vaccine development;
• synthesis of virus-like particles (VLP); 
• or to express the protein which has failed in mammalian expression system.​

Service highlight:
1. Team members have average 10 years’ experience in Baculovirus-insect expression system, 99% success rate.
2. Multiple expression cell lines and vectors available.
3. Up to 200L fermentation volume, possible reach 100g protein production.

Service and Timeline	Service Description	Deliverables	Price
P110411 Insect-Ready 5 - 7 Weeks	1. P1 generation virus production (low titer). 2. P2 generation virus production (high titer). 3. Small volume cell culture and expression test. 4. Protein purification (1-step, affinity purification). 5. SDS-PAGE test. 6. QC and report. Addition: > Gene codon optimization, synthesis and reconstructed plasmid.	1. 0.2mg-1mg purified protein. 2. Data and QC report.	Inquiry​
P110412 Insect-StepUp 9 – 11 Weeks	1. Condon optimization and gene synthesis (1000bp free). 2. P1 generation virus production (low titer). 3. P2 generation virus production (high titer). 4. Midi volume (XXX mL) cell culture and expression condition test: up to 3 different conditions. 5. Protein purification (1-step, affinity purification). 6. WB and SDS-PAGE test. 7. QC and report.	1. 0.5mg-5mg purified protein. 2. Data and QC report.	Inquiry​
P110413 Insect-Comfo 9 – 11 Weeks	1. Condon optimization and gene synthesis (free of charge). 2. 2 expression vectors will be used for reconstructed plasmid. 3. 2 cell lines will be used to test expression. 3. P1 generation virus production (low titer) and expression test. 4. P2 generation virus production (high titer) and expression test. 5. 200mL - 1L cell culture. 6. Protein purification with 2 steps, purity polishing to >85%. 7. WB and SDS-PAGE test. 8. QC and report.	1. 1mg - 10mg purified protein. 2. Data and QC report.	Inquiry​
P110402 1L Cell Culture 2 Weeks	1L insect cell culture and protein purification for 1 clone.	Up to 30mg purified protein.	Inquiry​

 

Optional Service 1. Tag remove 2. 5L~200L fermentation 3. Western Blot for protein confirmation 4. Mass Spectrometry More information: Please Email to info@bio-bench.com if you need special service for Baculovirus-insect Expression System.

List of Vectors	Commercial vectors and Bio Bench improved vectors. Please inquire for the up-to-date list.
List of Expression Strain	Sf9, Sf21, Hi5. Please inquire for the up-to-date list.

Comparison of different types of service

Service type Content	Insect cell Ready	Insect cell StepUp	Insect cell Comfo
Codon optimization	-	✓	✓
Gene synthesis	-	✓	✓
Multiple vector or multiple cell line test	-	-	✓
Expression temperature test: HighT °C	✓	✓	✓
P1 generation virus	✓	✓	✓
P2 generation virus	-	✓	✓
Expression duration test (time points)	1	2	2
Fermentation volume	80mL	200mL	200mL – 1L
Affinity purification	✓	✓	✓
2nd purification: purity enhancement	-	-	✓
SDS-PAGE analysis	✓	✓	✓
WB analysis	-	✓	✓
Data, QC and report	✓	✓	✓
Video meeting support	-	-	✓
Purified protein	0.3mg – 2mg	0.5mg – 5mg	1mg – 10mg
	Inquiry​	Inquiry​	Inquiry​

 

Advantages of Baculovirus-Insect Protein Expression System

1. Large gene capacity: ability to carry large gene fragment and express the protein, protein at 90kDa – 250kDa is recommended to be expressed by insect cell;
   
2. Multiple genes expression capacity: A high capacity for multiple genes or a large insert, because of the huge and flexible viral genome (130 kb);
   
3. Proper post-translational modification (PTM): insect cells are higher eukaryotes and able to modify the protein, particularly the glycosylation;
   
4. High biosafety: baculovirus has strict species specificity and naturally does not infect human;
   
5. High expression efficiency: high yield boosted by the strong promoters (polyhedron, p10, etc.), as well as high titers up to 10^8 pfu in late-stage infected cells enable large-scale protein expression;
   
6. High scale expression volume: Easy to amplify and can produce recombinant proteins in large scale, up to XXX L.

 

Case Studies - Baculovirus-Insect Protein Expression System

Case study 1 - Virus protein

A virus protein was successfully expressed by Sf9 cell line at 60mg/L. After purification, protein purity reaches 95%.
M: protein marker; R: reduced protein; N-R: non-reduced protein.

Case study 2 - Enzyme protein and bioactivity test

An enzyme protein was successfully expressed by Sf9 cell line. After purification, protein purity reaches 90%. Then enzyme activity was tested by reagent kit and the signal was read at OD520. The enzyme activity is 420U/mg after testing.
Fig. A - M: protein marker; 1: Elution by 250 mM imidazole; 2: Elution by 60 mM imidazole.
Fig. B – Standard curve of enzyme activity, R^2=0.9982.

Case study 3 - Cell signaling pathway protein expression and scale up production (5L)

PT protein is a cell signaling pathway protein. 1g of PT is needed by a client for his extended study. After trying in both CHO and HEK293 cell line, PT protein failed to achieve high expression level and 1g of protein may result in very high cost.
After analyzing the PT protein sequence, Bio Bench advised to go with baculovirus-insect expression system and later on with scale up production - purification.  The PT protein was highly expressed by both Sf9 and Hi5 at 230mg/L and 280mg/L, respectively. Then reconstructed Hi5-PT was then cultured at 5L volume following by affinity purification. 1.24g purified protein (purity > 95%) was harvested and delivered to client’s lab bench.

Step 1 – P1 generation virus infection and expression test.
Reconstructed bacmid infected both Sf9 and Hi5 cell line. The protein was successfully secreted to culture medium as well as soluble Intracellular expression (result only show Sf9). The intracellular soluble fraction was selected by client for further study.
M: protein marker; Φ: non-infected Sf9 cell; 1 and 2: clone 1 and clone 2 both expressed by Sf9 cell; Culture medium: protein expression in culture medium; Soluble protein: soluble expression in intracellular; Insoluble protein: insoluble expression in intracellular.

Step 2 – P2 generation virus MOI test
Virus was collected and infected Sf9 and Hi5 cells, separately. Different MOIs (a, b, c in the images above) were applied during the test to find out the best MOI. 2 different time points (D-A, D-B) fractions were analyzed. Protein expression level: Sf9 is 230mg/L and Hi5 is 280mg/L.
M: protein marker; Φ: non-infected Sf9 cell and Hi5 cell; D-A and D-B: cell culture days; a, b and c: different MOI.

Step 3 – 5L scale up production and purification and QC
The best conditions were applied and 5L Hi5 cells were cultured for scale up production. Affinity purification method was used to harvest PT protein. Protein quantity was tested by Bradford method and purity tested by SDS-PAGE. Totally 1.24g protein was harvested and purity > 95%.
M: protein marker; IN: injected cell lysate; FT: flow through; W1 – W3: wash fractions; E1 – E9: elution fractions.