Cell wall synthesis protein KRE9-producing Pichia pastoris cell line

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Catalogue Number	160659
Antigen/Gene or Protein Targets	KRE9
Parental Line	Pichia pastoris cells
Host	Yeast
Model	Over-expressing
Research Area	Microbiology
Recommended Growing Conditions	YPD broth containing 100 μg/ml ZeocinTM at 28-30°C and 250 rpm of shaking
Notes	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 30°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 30°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 μm diameter pore membrane, concentrated by a tangential concentrator using an ultrafiltration membrane, and stored at -80°C until further use. Recombinant proteins were precipitated from the concentrated supernatant with ammonium sulfate.

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Andoni Ramirez Garcia

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	University of the Basque Country, EHU
	13 reagents listed on Ximbio

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