pPICZαA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The yeast expression library vectors were linearized by SacI digestion and transformed into P . pastoris X-33 with the lithium chloride transformation method described in EasySelectTM Pichia expression kit user manual (Invitrogen). Transformants were initially grown on YPD plates supplemented with 100 µg/ml zeocin. After integration of the plasmid into the yeast genome was confirmed by colony PCR, resulting colonies were transferred to YPD plates with 200, 500 and 1000 µg/ml zeocin for determination of the copy number of integrants.

Protocol tips

The yeast expression library vectors were linearized by SacI digestion and transformed into P . pastoris X-33 with the lithium chloride transformation method described in EasySelectTM Pichia expression kit user manual (Invitrogen). Transformants were initially grown on YPD plates supplemented with 100 µg/ml zeocin. After integration of the plasmid into the yeast genome was confirmed by colony PCR, resulting colonies were transferred to YPD plates with 200, 500 and 1000 µg/ml zeocin for determination of the copy number of integrants.

Publication protocol

"The yeast expression library vectors were linearized by SacI digestion and transformed into P . pastoris X-33 with the lithium chloride transformation method described in EasySelectTM Pichia expression kit user manual (Invitrogen). Transformants were initially grown on YPD plates supplemented with 100 µg/ml zeocin. After integration of the plasmid into the yeast genome was confirmed by colony PCR, resulting colonies were transferred to YPD plates with 200, 500 and 1000 µg/ml zeocin for determination of the copy number of integrants. The subsequent comparisons of secreted proteins were only made between transformants with approximately the same copy numbers as determined by the same concentration range of drug resistance against zeocin.
The experimental protocol from EasySelectTM Pichia expression kit user manual to express recombinant Pichia pastorish has been followed. Briefly, the selected colonies were initially cultivated (shaking vigorously at 250 rpm) in BMGY medium at 28-30°C until the value of OD600 reached 2 approximately. After centrifugation and removal of BMGY, cell pellets were re-suspended in BMMY to an OD600 of 1 to induce expression. The volume of the culture should be no more than 10-30% of the total well/tube/flask volume to ensure sufficient aeration. Methanol was added to a final concentration of 1% every 24 hours to maintain induction. Yeast culture media were sampled and assayed every 24 hours. Small-scale cultivation and expression using 96-deep-well plates (Bel-Art Scienceware, NJ, USA) were carried out as previously described [42,43] whenever high-throughput screening of the secretory productivity was needed."

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 SCF using pPICZαA from Donghai Wu, The Key Laboratory of Regenerative Biology and The G.

Manufacturer protocol

Download the product protocol from Donghai Wu, The Key Laboratory of Regenerative Biology and The G for pPICZαA below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - P. pastoris SCF using pPICZαA from Donghai Wu, The Key Laboratory of Regenerative Biology and The G. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.