pwPICZalpha-DT390-bi-pIL-2-Non-N-Gly

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Protein expression and purification in Pichia pastoris were performed as previously described with the following modifications (Wang et al., 2011; Peraino et al., 2012).	A Ni-Sepharose fast flow resin (GE healthcare) was used for the purification. Porcine IL-2 fusion toxins were eluted using 40 mM imidazole. Western blot analysis, FACS analysis, FACS competition/blocking analysis and KD determination were all performed as previously described (Peraino et al., 2012) using LCL13271 cells (Cho et al., 2007).

Protocol tips
Protein expression and purification in Pichia pastoris were performed as previously described with the following modifications (Wang et al., 2011; Peraino et al., 2012).
Downstream tips
A Ni-Sepharose fast flow resin (GE healthcare) was used for the purification. Porcine IL-2 fusion toxins were eluted using 40 mM imidazole. Western blot analysis, FACS analysis, FACS competition/blocking analysis and KD determination were all performed as previously described (Peraino et al., 2012) using LCL13271 cells (Cho et al., 2007).

Publication protocol

Protein expression and purification in Pichia pastoris were performed as previously described with the following modifications (Wang et al., 2011; Peraino et al., 2012). A Ni-Sepharose fast flow resin (GE healthcare) was used for the purification. Porcine IL-2 fusion toxins were eluted using 40 mM imidazole. Western blot analysis, FACS analysis, FACS competition/blocking analysis and KD determination were all performed as previously described (Peraino et al., 2012) using LCL13271 cells (Cho et al., 2007). The Ontak®-like monovalent human IL-2 fusion toxin (DT390-hIL-2) used as a control for our in vitro assay was constructed, expressed and purified exactly same as the monovalent porcine IL-2 fusion toxin. The DT390 alone and the non-N-glycosylated porcine IL-2 alone (pIL-2-Non-N-Gly) were used as controls for our in vitro assay. These products were also expressed and purified in the yeast Pichia Pastoris system.

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Download the product protocol from Zhirui Wang, Transplantation Biology Research Center, Massachuse for pwPICZalpha-DT390-bi-pIL-2-Non-N-Gly below.

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