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Protein expression in Pichia pastoris X-33 strain

Protein expression Pichia Xylosidase Protein amount

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#1 Boopathi



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Posted 26 August 2011 - 01:37 AM

Dear All

We are using Pichia X-33 wild type strain to express our target protein. The vector pPICZalphaA was used to transform the target gene by EasyComp method from Invitrogen. Initially the target gene was inserted into the circular plasmid and inserted into Ecoli BL21 and linearized with BstXI. The zeocin resistant transformants were selected on YPDS with Zeocin plates. Then we tested the Mut+ phenotype in Minimal Methanol medium and Minimal Dextrose medium. The cells grown faster in MD medium than in MM medium eventhough the X-33 strain is Mut+ phenotype. After this inorder to check the protein expression, a single colony of the clone was grown on Buffered Minimal Glycerol medium and Minimal Glycerol Medium. The incubation conditions were 30C and 250rpm. The invitrogen protocol recommended the cells to be used for methanol induction in the OD range of 2-6 after 16-18 hours. But the OD never reached above 1.5 after 18 hours to 48 hours. We used the cells to Methanol induction and took sample every 12 hours upto 96 hours. Since we used the vector with alpha factor, the target protein should be secreted in the supernatant. But there is no detectable amount of protein present in the supernatant. Please tell us where the problem occur, why the X-33 cannot utilize the methanol and why there is no protein secreted in the medium.

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