Protein production and purification in Baculovirus / Insect cells

Custom recombinant protein expression and purification in sf9, sf21 and High five (Hi5) cells

Contact us Our state of the art Insect cells / Baculovirus recombinant protein production platform enables ProteoGenix to offer the highest quality of services of production of proteins through proprietary and optimized step by step protocols:

Step 0: Gene synthesis and codons optimization

Proteins production gelThis gene synthesis step is very important because it allows us to optimize rare codons for baculovirus expression system. In many cases it increases production yields very significantly.

Step I: cDNA cloning into an expression vector

This step consists in integrating the cDNA into a baculovirus expression vector containing a tag and inducing the expression of the fusion protein of interest and allowing the protein purification on affinity columns via the tag. It is possible to cleave the tag when necessary especially when protein activity is necessary.

Step II: Recombinant virus generation and recombinant protein expression evaluation in baculovirus

  • Generation of recombinant bacmid DNA
  • Transfection of insect cells with recombinant bacmid
  • Generation of virus stocks
  • Infection of insect cells
  • Quality control by acrylamide gel (SDS-PAGE) and / or Western blot

Step III: Expression and purification scale up from 1 Liter:

  • Expression of protein and purification from one or more liters of insect cell culture
  • Affinity purification run(s) on column
  • Quality control by acrylamide gel (SDS-PAGE) and / or Western blot

Turnaround time:

  • Cloning: 2 weeks
  • Expression evaluation: 5 to 6 weeks
  • Production and purification: 1 to 2 weeks

Baculovirus (Insect cells) system technical overview

Baculoviruses are a family of viruses featured by a rod-shaped appearance (in Latin Baculum means stick), the Baculoviridae. They are characterized by two sub-families: Eubaculovirinae with Nucleopolyhedrovirus (NPV) and Granulovirus (GV), the Nudibaculoviridae. These viruses can infect mostly moth species but they have also been found infecting sawflies, mosquitoes and even crustaceans.
Today, no known baculovirus is capable of infecting mammals or other vertebrates. The baculovirus genome consists of a double-stranded circular DNA whose size is between 80 and 180 kbp, which makes it a large virus.
Baculoviruses are used in biotechnology for the production of recombinant proteins since 1983. These viruses enable to introduce the gene encoding the protein to be produced in cultured insect cells, including cells of Spodoptera frugiperda (Sf9 and Sf21) and Trichoplusia ni (Hi5 or High five)
The cDNA of the protein of interest is integrated into the genome of the virus by replacing the gene encoding the fibril structure protein p10, which is not useful for the virus in cell culture. The protein interferon beta is an example of a protein produced in baculovirus.

ProteoGenix’ baculovirus protein production platform

ProteoGenix offers services of synthesis of proteins in baculovirus / insect cells Sf9, Sf21 or Hi5. The advantage of this system (Baculovirus Expression Vector System : BEVS) is that after a production and purification you get a functional recombinant protein with correct post translational modifications (PTM): disulfide bonds formation, glycolsylation (N-and O- ), signal peptide cleavage, no N-terminal methionine, myristylations, acetylations, phosphorylations, ubiquitinations, glycolipid anchors, multiple splicing, heterodimeric assemblies, pro-peptides maturation, etc. These modifications guarantee a good biological activity. In many cases BEV system is preferred from other eucaryotic systems like mammalian cells for intra cellular expression.

Protein development in insect cells and post translational modifications (PTM)

Some post-translational modifications (PTM) such as myristylations, acetylations, phosphorylations, ubiquitinations and glycolsylations (O-and N-) are correctly performed only in complex eukaryotic organisms. Baculovirus system was not perfect until very recently for the production of mammalian proteins, in the sense that some types of insect cells were not able to make N-glycosylation entirely (It only reached the 3 mannose molecules), although they are at the right place. However, Sf9 cells for instance, can now produce N-glycosylation similar to those produced in mammalian cells (the only difference being that the sialic acid is modified by a hydroxyl).

Insect cells expression system advantages

The amounts of protein obtained from our baculovirus system are generally greater than with any other advanced eukaryotic system and especially for intracellular proteins. Other advantages of the baculovirus / insect cell (insect larvae) system are genomic stability and the safety aspect in that cells are grown without animal proteins.