Hi everyone! I'm reading a lot on yeast expression system for heterologous expression. I was wondering if it was possible to get some insights on challenges with this system and good troubleshooting plans to go with it?
Hi Susan,
the main problem in yeast expression is to crash the cells to extract your expressed protein (yeast has a robust cell wall, it is not enough a sonicator). To overcome this problem you can add a secretion pro-peptide that allows you to harvest the expressed protein in the medium after induction. In my experience this approach works modestly, I suspect mainly because the protein get stuck in the golgi where is heavily modified. I managed to get small proteins (20 to 50 kD) in the medium but not big ones. So it mainly depends on the nature of the protein you want to express. I got very good results with big proteins but you need to plan for the protein extraction. I use 5 mm glass beads that I add to tubes with induced cells and the Fastprep machine that shakes the cells vigorously until all protein content is released in the buffer. This works well and you can express your proteins as GST-, MBP-, 6His-, Streptag- or ChitinBindingDomain- or with HA or Myc tags to purify with antibodies. Hope this helps.
M
It would help if you could tell which yeast system your are considering. There are a wide range of secretion tags available for secretion of your protein into the medium, such as alpha-factor for S. cerevisiae, Ost1 signal for P. pastors or even Lip2 signal for Y. lipolytica. All these tags have their limitations depending on the nature of the protein you are trying to express as sometimes they can interact with the protein or form aggregates, hampering secretion. If you don't intend on secreting the protein then cell lysis using glass beads is the common practice. S. cerevisiae has high copy number expression plasmids available so the expression would be greater in this system if that's what you are looking for.
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