pPICZαB/α-amylase

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Transformation of the gene of interest into P. pastoris GS115 strains was achieved by electroporation method. The electrocompetent cell was prepared according to EasySelect Pichia expression kit manual with minor modifications.

Protocol tips
Transformation of the gene of interest into P. pastoris GS115 strains was achieved by electroporation method. The electrocompetent cell was prepared according to EasySelect Pichia expression kit manual with minor modifications.

Publication protocol

Transformation of the gene of interest into P. pastoris GS115 strains was achieved by electroporation method. The electrocompetent cell was prepared according to EasySelect Pichia expression kit manual with minor modifications. The recombinant plasmid pPICZαB/α-amylase was linearized by using SacI restriction endonuclease prior to gene integration into the P. pastoris genome. Transformants were selected on YPDS agar plates containing yeast extract (1% w/v), peptone (2% w/v), dextrose (2% w/v), 1 M sorbitol, and agar (2% w/v) supplemented with 100 μg mL−1 zeocin at 30°C. Single colony of P. pastoris grown on YPDS was inoculated into 10 mL YPD broth and incubated overnight at 30°C under shaking conditions at 250 rpm. Then, 500 μL of the culture was inoculated/transferred to a 500 mL volume DURAN Erlenmeyer flasks with baffles (DURAN Produktions GmbH & Co. KG, Mainz, Germany) containing 100 mL of BMGY and incubated in a shaking incubator at 30°C for 24 h at 250 rpm. The cells were harvested and adjusted to OD600 nm = 10 in 50 mL of BMMY medium. The culture was induced with methanol (0.5% v/v) for 48 h with a 24 h interval. One mL of the culture was harvested, centrifuged at 3000 ×g for 10 min at 4°C and the supernatant was subjected to α-amylase assay using the DNS method with minor modifications [18].

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Eukaryotic cells - P. pastoris G. stearothermophilus SR74 α-amylase using pPICZαB/α-amylase from Siti Nurbaya Oslan, Department of Biochemistry, Faculty of Biote.

Manufacturer protocol

Download the product protocol from Siti Nurbaya Oslan, Department of Biochemistry, Faculty of Biote for pPICZαB/α-amylase below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - P. pastoris G. stearothermophilus SR74 α-amylase using pPICZαB/α-amylase from Siti Nurbaya Oslan, Department of Biochemistry, Faculty of Biote. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.