Baculovirus Expression

Proteos offers baculovirus mediated protein expression in our fully licensed Sf9 (Spodoptera frugiperda) and Tni PRO (Trichoplusia ni) cell lines.  Both cells lines are suitable for the production of intracellular or secreted recombinant proteins with a majority of the post-translational modifications found in mammalian cells. The Tni PRO cell line has been shown to produce higher levels of secreted protein expression in many cases.

The baculovirus expression vector systems (BEVS) uses homologous recombination or site-specific transposition to produce recombinant virus encoding the protein of interest. To ensure accurate MOIs (multiplicity of infection) and reproducibility of each production run, Proteos utilizes a robust plate-based flow cytometry assay for determining the titer of baculovirus stocks prior to infection of insect cells for protein production.  The titer of each virus stock that we produce is determined in triplicate using our proprietary plate-based flow cytometry assay.  We routinely produce high-titer virus stocks with titers ranging from 5 x 108 – 2 x 109 infection units (IU) per milliliter in a single amplification.

Learn More about Baculovirus Titer Assay Services

We utilize optimized and fully validated insect cell expression protocols for consistent reproducible high-level protein expression in serum-free shake flask culture volumes up to 40 L per batch. Only mycoplasma-free, high viability, low passage cultures are used for expression and are monitored using a Beckman Coulter Vi-CELL TM XR for culture density, viability, and average cell diameter.  The Vi-CELL TM XR uses the trypan blue dye exclusion method and averages 50 measurements to compute the parameters for each sample.  This allows us to monitor cultures with a high degree of accuracy which is imperative for quality and reproducibility of protein production projects.  All cell culture data is included in a customized technical report delivered with each project.

Typical productions times are 4-6 weeks for construct design, expression, and purification.  

Services offered:

  • Proteos offers a full suite of molecular biology services to accompany any of our expression platforms.  Molecular biology services available include construct design, fusion partner and affinity tag selection, gene synthesis with codon optimization, subcloning, endotoxin-free plasmid isolation, and sequence verification.
  • Small-scale expression verification at less than 100 mL scale, ideal for expression testing of multiple recombinant protein targets.
  • MOI (multiplicity of infection), cell line, and time of harvest optimization
  • Analytical production and yield determination at scales less than 2 L, ideal for process verification through purification prior to large-scale production.
  • Large-scale recombinant protein production up to 40 L per batch in shake flasks.
  • Generation, cryopreservation, and banking of BIIC (baculovirus infected insect cells) stocks for rapid scale-up and expression in future production runs
  • Concentration of culture supernatant via tangential flow filtration and buffer-exchange diafiltration for secreted His-tag proteins to be purified by Ni-NTA chromatography
  • Titer determination of high-titer baculovirus stocks using Proteos’ proprietary flow cytometry assay
  • Delivery of cell paste, conditioned culture supernatant, concentrated buffer-exchanged culture supernatant, high titer baculovirus stocks, cryopreserved BIIC stocks
  • Insect cell expression services can be combined with Proteos’ purification services for a fully-customized production process.