Problems Expressing from Kluyveromyces lactis - (Jun/21/2011 )
I need help with a construct I'm trying to express in Kluyveromyces lactis (GG799 strain). Last year one student managed to transform this construct (coding for a fusion protein of about 20 kDa). After making patches of the candidate colonies (integration verified by three different PCR reactions), 2 ml cultures of YPGal were grown for 24, 48 and 72 h. To our surprise we could see a band of the right size already at 24 h by Coomassie staining of the crude supernatant (25 ul loaded in the gel). Unfortunately, these cells got contaminated with bacteria before he could make glycerol stocks to keep. This student is not in the lab any more, and I have tried to repeat the experiment starting from the transformation of yeast. I have many positive candidate colonies (by PCR), but none of them express the protein. I cannot imagine what I am doing different from what he did. Anybody has had this happened? Is there something on the medium that needs to be very well controlled? Any suggestions? Thank you!!
I may not be able to help you regarding this problem as i'm having the similar situation as you are (no protein secreted) . I just want to ask you about your integration PCR product. Did you get the 2.4 kb/ 2.3 kb band for positive transformant? In my case the bands are 2.6 kb and only control protein (MBP)was expressed.
Regarding the medium, my senior mentioned about the pH need to be strictly controlled but i'm not sure if it really necessary. By the way, have you done western blot?