CELL LINES
Contributor Information
- Name Andoni Ramirez Garcia
- Institute University of the Basque Country (EHU)
Tool Details
- Tool name: Cell wall synthesis protein KRE9-producing Pichia pastoris cell line
- Tool type: Cell Lines
- Parental cell line: Pichia pastoris cells
- Organism: Yeast
- Model: Over-expressing
- Production details: For expression of recombinant protein: P. pastoris cells containing the gene of interest were plated in YPD plus ZeocinTM agar plates and single colonies were grown in 25 ml of Buffered Glycerol-complex Medium (BMGY; 1% yeast extract, 2% peptone, 100 mM PBS (pH 6), 1.34% yeast nitrogen base (YNB), 4 x 10-5% biotin and 1% glycerol) at 30ÄÂ?°C and 250 rpm until OD600=2-6 was reached, for about 16-18 h. The pellet was collected by centrifugation and diluted to OD600=1 in Buffered Methanol-complex Medium (BMMY; prepared as BMGY, but with 0.5% methanol instead of glycerol) and incubated at 30ÄÂ?°C and 250 rpm for 72 h. Every 24 h, methanol to a final concentration of 0.5% was added in order to maintain a constant induction of the expression of the protein. Finally, samples were centrifuged and the supernatant was filtered by a 0.22 ÄÂÄ?m diameter pore membrane, concentrated by a tangential concentrator using an ultrafiltration membrane, and stored at -80ÄÂ?°C until further use. Recombinant proteins were precipitated from the concentrated supernatant with ammonium sulfate.
- For Research Use Only
Target Details
- Target: KRE9
Application Details
Handling
- Format: Frozen
- Growth medium: YPD broth containing 100 ÄÂÄ?g/ml ZeocinTM at 28-30ÄÂ?°C and 250 rpm of shaking
- Shipping conditions: Dry ice
Documentation
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