The baculovirus-insect cell expression system is widely used to produce recombinant proteins for many different biomedical applications. It provides researchers with the means to generate milligram quantities of recombinant proteins that are ideal for functional and structural analysis. The system typically produces overexpressed recombinant proteins with proper folding, disulfide bond formation and oligomerization. In addition, proteins produced with this system are with proper post-translational modifications, including N- and O-linked glycosylation, phosphorylation, acetylation, amidation, methylation, isoprenylation, signal peptide cleavage and proteolytic cleavage. The sites where these modifications occur are often identical to those of the authentic protein in its native cellular environment. Baculovirus-expressed recombinant proteins thus resemble the native version in functionality.

 

Phase/Description

1) Molecular Biology

  • subcloning your gene of interest into a baculovirus transfer vector
  • bacmid DNA preparation and verification by PCR

2) Producing Recombinant Baculovirus and Titer Determination

  • transfecting insect cells
  • isolating P1 Viral Stock (low-titer stock)
  • generation of high-titer stock in cells from low-titer stock
  • determination of the titer by end-point dilution assay

3) Expression Optimization

  • time course infection of log phase cells (Sf9) using inoculum from generated and titered high-titer stock (MOI =0.2-10 variables)
  • maintenance of time course infections with supernatants harvested between 48-96 hours post infection
  • samples from different time point, including cell and supernatants, will be sent to the customer for time point detemination for recombinant protein expression.

4) Large Scale Expression

  • one liter of insect cells will be infected with high-titer virus stocks
  • two to four days after transfection, cells and supernatant will be harvested, frozen and shipped to the customer

5) Recombinant Protein Purification

  • protein purification using a Ni-NTA (for 6xHIS-tagged proteins) column at either native or denatured condition.

 

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