3 replies to this topic

[sean]

hi.

I have had some problems with e coli expression/purification, so i decided to change my system entirely.  ( maybe induction was too high, all my proteins ended up in inclusion bodies and had to do GuHCl isolation but proteins crashed out during refolding etc...  it was a mess...)

I'm gettin set up for expression in yeast.  Does anyone have any expriences in protein expression/purification in yeast( strain DY150)??  main concerns i have the high proteloysis in yeast.

and havent decided which method i'm gonna use to crack the cells....   If someone has some info and reference, i'll appreciate it.

sean

Much obliged.

[ragno]

Hi,
Do you have tried induction with low IPTG concentration and/or low induction T° (e.g 30°C) ? In my labo, a friend has made a success to obtain a protein soluble with a induction at 20°C!!!
You can also to try a cells lysis with sulfobetain, you can obtain a soluble protein fraction from inclusions bodies.
Then, for yeast expression, i have made this with pichia pastoris, there are yeast expression manuals with good protocols and hints.
good luke
michel

[kathia]

Hello,

My question is for RANGO, you said in your answer that in your lab  use IPTG to express in yeast, so what medium and vector did you use?. Do you use minimal medium? I have a lot of problems, on my gels I don't see clear bands. Do you have protocols?, could you send me it by email, please I'll really apreciate. my email is: kathia_33@hotmail.com. OK if anyone can help me please contact me.

[jadefalcon]

Hi there!

Unfortunately I don't have something to say about yeast, but  maybe I can bring your attention to yet another expression system ...

What I wanted to say is, that we had a similar problem some time ago and we went to express our protein in Drosophlia Schneider-2 cells in FCS-free medium.

Pros: Directly inducible by Cu2+ ions in the medium, since the GOI is under the regulation of a metallothioneine promotor, AND we got our protein secreted into the cell culture media, since our expression construct had a secretion-sequence, too. So, no need to crack or lyse cells, just a centrifugation, and there you have your precleared protein in suspension....    
AND, if you're doing immunology with your protein, you have the guarantee that it's free of stimulatory prokaryotic factors such as bacterial DNA, or LPS etc.

Cons: You need some time to establish your stably transfected cell line,  optimal induction times can vary from protein to protein (7 days in my case) AND your gene has to be expressed by DS-2 cells at all (codon bias, splicing etc.)

But when it works, it's a very fine thing indeed!

@katia: I think ragno was talking about expression E.coli and different induction temperatures...

Mike

Edited by jadefalcon, 27 July 2004 - 04:38 AM.