pPIC9K-duIL-2

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	The recombinant plasmid pPIC9K-duIL-2 was linearized with SalI and 10 µg of purified DNA fragment of linearized plasmid pPIC9KduIL-2 was transformed into the competent P. pastoris GS115 cells by electroporation in a Biorad GenePulser with settings of 1,800 V, 25 µF capacitance, and 400 ohms resistance. Selective culture medium without histamine (MD/His-) was performed to check the yeast transformants.

Protocol tips

The recombinant plasmid pPIC9K-duIL-2 was linearized with SalI and 10 µg of purified DNA fragment of linearized plasmid pPIC9KduIL-2 was transformed into the competent P. pastoris GS115 cells by electroporation in a Biorad GenePulser with settings of 1,800 V, 25 µF capacitance, and 400 ohms resistance. Selective culture medium without histamine (MD/His-) was performed to check the yeast transformants.

Publication protocol

"The recombinant plasmid pPIC9K-duIL-2 was linearized with SalI and 10 µg of purified DNA fragment of linearized plasmid pPIC9KduIL-2 was transformed into the competent P. pastoris GS115 cells by electroporation in a Biorad GenePulser with settings of 1,800 V, 25 µF capacitance, and 400 ohms resistance. Selective culture medium without histamine (MD/His-) was performed to check the yeast transformants. The His+ transformants were inoculated to the corresponding positions of YPD plates with G418 of various concentrations (0, 0.5, 1.0, 1.5, 2.0 g/l), respectively, and incubated at 30oC for 2-3 days. The well-grown strains with high G418 resistance were screened out. Then the genomic DNA of the yeast
transformants was extracted, and PCR with duIL-2 primers was applied to examine the right transformants. The correct recombinant yeasts were cultured in BMGY (50 ml/250 ml flask) and cultured at 30oC to an OD600 value of about 3.0. Yeast strains were collected by
centrifugation and resuspended in BMMY medium (20 ml/150 ml flask), and then induced for expression with methanol [1% (v/v)] for 5 days. The suspension of 1.0 ml was sampled every day. The supernatant of the suspension was separated by centrifugation and
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)."

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