Use the Pichia secreted expression system - White ppt formed in the supernatant after stored at -80 C fridge (Mar/12/2006 )
Dear All,
I m working on the Pichia expression system. I cloned my target gene into the pPICZ-alpha-A vector(the recombinant protein will be secreted into the medium when use this vector) and tested the expression. I collected the culture and then spined down the cells and kept the supernatant. Actually, the supernatant was very clear after centrifugation. I kept the supernatant at -80 C for further analysis. However, when i took it back from the -80 C fridge, I thawed the supernatant and I found there were white ppt formed. I don't know what these white ppt are. Anyone body has this experience?
Thx very much!!!!!
siuchi
Might be some precipitated proteins. I´ve used the same expression method and Pichia also secrete a lot of native proteins. I didn´t saw white ppt, but I did my experiments on the fresly isolated medium.
Dear aspergillie,
Thx very much!
I would like to ask more about your protein expression using Pichia system. Could you get significant amount of purified protein eventually? What kind of purification system you use?
Thx again!!!
siuchi
Hi siuchi98,
A white precipitate once formed in some Pichia culture medium stored overnight at 4 degrees. I collected some of it and ran it on an SDS-PAGE gel, but there were no proteins there. Maybe sugars coming off the glycosylated proteins? I don't really know what that stuff was. I didn't pursue this problem further.
I've never isolated the protein. I expressed proteins fused with YFP and CFP for FRET experiments. At that time, if I had to isolate it, I would have done it by binding antibodies to a column, but of course there are many other ways which might be better for you.