Use the Pichia secreted expression system - White ppt formed in the supernatant after stored at -80 C fridge (Mar/12/2006 )
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!!!!!
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.
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?
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.