Technology

About Silkworm-Baculovirus
 Expression System

Technology Overview

About Silkworm-Baculovirus Expression System

ProCube offers the silkworm-baculovirus expression system as a viable alternative to protein expressed in E. coli, yeast or other baculovirus expression systems. Utilizing a natural viral infection for silkworms (baculovirus), we leverage every cell in the silkworm’s body (excluding the head and abdominal cavity) to rapidly produce structurally complex and biologically active protein. Universally recognized as a powerful and versatile system for production of high quality proteins, the baculovirus expression system has several advantages over E. coli such as improved solubility, ability to incorporate post-translational modifications, and higher yields for secreted proteins.

Silkworms are considered highly-dense disposable bioreactors that do not require aseptic conditions to operate. Since Maeda et al. (1985), reported the production of human alpha interferon in silkworm larvae, using recombinant B. mori nucleopolyhedrovirus (BmNPV), the production of thousands of proteins have been achieved1. And since the baculovirus does not infect humans, another advantage is there are no biohazards associated with its use for laboratory workers. Likewise, since silkworms are domesticated and the moths cannot fly, the biosafety risk is very low for the environment. Expression levels are 10- to 100-fold higher than using Bombix mori (silkworm) cell line or conventional insect cell lines (e.g., Sf cells)2. And only a small amount of recombinant baculovirus is needed.

Protein Production

Step1
Construction of the Transfer Vector
ProCube takes customer’s gene of interest (GOI), either as a plasmid or gene fragment, and constructs a transfer vector with customer’s GOI inserted.

Step2
Co-transfection
The recombinant transfer vector (recombinant plasmid) is co-transfected with the baculovirus DNA into the BmN cells. Recombinant viruses appear after six days in the media.

Step3
Production of the Recombinant Protein
ProCube uses a sterile syringe to manually inject a larva or pupa. ProCube harvests the hemolymph (larva) or tissue (pupa) after six days incubation. Then we filter and centrifuge the extract in order to obtain soluble fractions. After separation we purify our customer’s target protein using a variety of standard chromatography columns.

1 Maeda et al., (1985). Production of human alpha-interferon in silkworm using baculovirus vector. Nature. 315(6020):592-4

2 Hiyoshi et al., (2007). Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein. J Virol Methods. 144(1-2):91-7

Advantages of Silkworm-Baculovirus Expression System

We match the strength of recombinant technology with the power of living insect host to produce high quality protein. We express protein using a living silkworm as a host expression system. This in vivo technique offers several distinct advantages over traditional insect cell culture systems.

Express Your Protein In vivo

■ Difficult proteins with higher yield
The silkworm offers a cellular environment that is naturally heterogeneous which offers a higher probability of expressing than traditional homogeneous insect cell culture. Posttranslational modifications are similar to mammalian cells. Coupled with our patented cysteine protease gene defective (CPd) baculovirus that inhibits fragmentation of target protein and increases yield three times more than wild type baculovirus, the silkworm overexpresses difficult proteins, such as GPCRs and multi-subunit proteins easily.

Multi-parallel Protein Expression

■ We use hundreds of bioreactors
Silkworms are like very compact single-use bioreactors. Our protein production capacity is not limited by space or number of proteins, we utilize hundreds of bioreactors simultaneously. ProCube is ideal for processing lots of multiple proteins or many variants of the same protein target in parallel for structure-based drug discovery or HTS.

Scalable Production System

■ Reliably adjust to research needs in real-time
Once the yield per silkworm is determined, we scale linearly to meet your needs. We only need to increase the number of silkworms to reliably adjust to your scale. There is no need to adjust MOI like other baculovirus expression systems require.

Expedited Turnaround Time

■ We speed up your research
We accelerate your research by expressing proteins in only six days after infection. Our disposable bioreactors eliminate downtime required to sterilize bioreactors between customer orders. And since there are no major changes with upstream processing between Trial and Manufacturing phases, we can deliver your protein faster. Once we establish a protocol for your target protein, we can provide repeat orders in as little as two weeks.
Expression and Purification Examples