Catalog #25680 - Small scale expression scouting and optimization

Small scale expression scouting and optimization

Small scale expression scouting and optimization

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 ~1.0×108 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.

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, Curia is an authorized contract research manufacturer of pOET expression plasmids (Oxford Expression Technologies). Inquire for more information.

1. P1 Virus 2. Purified protein 3. Study Report 4. Certificate of Analysis

Material Required:
Plasmid harboring ORF for target protein

1-2 Weeks