The Baculovirus Expression System (BEVS) is an eukaryotic system that utilizes the Autographa californica nuclear polyhedrosis virus (AcNPV) for high-level expression of recombinant proteins in lepodoptera-derived insect cell lines, such as Sf9, Sf21, and High Five cells. Recombinant proteins produced using the system typically have correct folding, disulfide bond formation, and oligomerization, resulting in production of proteins that are functionally similar to their native forms. The system can accommodate large DNA inserts, perform intron/exon splicing, and yield relatively high levels of recombinant protein (0.1 to 50% of total protein). Short affinity tags, such as polyhistidine and Strep-tag II, are often fused to recombinant proteins to aid in detection and purification of proteins from BEVS.
In addition, baculovirus particles (BVPs) based ELISA to measure polyspecificity binding by therapeutic antibodies is a standard in vitro assay in assessing therapeutic antibody developability.
1. Target gene(s) are cloned into pFastBac expression plasmids (ThermoFisher) with N or C-terminal His and/or Strep tags.
2. P1 virus (~30 ml at ~1.0x108 pfu/ml) is generated from plasmid by transfection of bacmid DNA into Sf9 cells.
3. If long term storage of the virus is required, a BIIC (Baculovirus Infected Insect cell) stock can be prepared.
4. Small-scale expression analysis of the target protein is then analyzed by infecting two cell lines with two dilutions of virus and harvesting the cultures at two viabilities (8 data points). Affinity pulldowns are conducted from cell lysates or conditioned media and analyzed by PAGE and Western blot. The condition with the highest level of recombinant protein is identified.
5. 1 liter expression in insect cells is performed and harvested at the expression conditions identified in the small scale expression analysis. The protein is purified by affinity chromatography using nickel resin or tandem affinity chromatography using nickel and streptactin resins. Elution fractions are analyzed by PAGE & Western blot and pooled based on purity metrics established with the client.
1) Catalog No.Custom includes vector construction and P1 virus generation.
2) Catalog No.Custom includes small-scale expression analysis.
3) Catalog No.Custom includes 1L expression, and 1 step affinity purification.
4) Catalog No.Custom includes 1L expression, and tandem affinity purification (>90% purity is typically achieved but is not guaranteed).
5) Catalog No.Custom includes 1L expression, tandem affinity purification, and an SEC polishing step.
Please note: production may subsequently be scaled up to 10 liters or higher to produce protein quantities needed by clients.
Options available: low endotoxin processing, affinity tag removal by protease cleavage, protein titer quantification (HPLC), analytical protein characterization including a SEC-HPLC, C-IEF, thermal stability analysis, mass-spec, and protein formulation optimization.
As an alternate to the Bac to Bac system, Lake Pharma is an authorized contract research manufacturer of pOET expression plasmids (Oxford Expression Technologies). Inquire for more information.
Plasmid harboring ORF for target protein
1. P1 Virus
2. Purified protein
3. Study Report
4. Certificate of Analysis
5. For BVP production: 1 prep enriched baculovirus particles from Sf9 cells infected with empty virus, sufficient for 12 X 96 well plates.