I was expressing Membrane protein (100 KD plant cell wall protein with N-terminal His tag) in Saccharomyces Cerevisiae. The vector i am using is PDDGFP modified vector which have URA selection and GAL1 as promoter. I used URA media and Galactose (2%) for induction. I have expressed this construct several times but now for unknown reason it is not expressing. Using exactly the same protocol and reagent. What i already tried are1. Autoclaved as well as filtered media2. Autoclaved as well as filtered Galactose solution3. Fresh transformed cell line from -80C stock4. Newly transformed cell as well.5. Checked the Sequence again and was fine. Please suggest me. M. Obayed Ullah, PhD E.mail: [email protected] [email protected]
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