Probelm with expression of ~85 kDa GSt fusion protein. - (Nov/29/2007 )

Hello,

I have ben unable to get a good expression of my GSt fusion protein (~85 kDa). I have tried expressing under different conditions namely; Induction at 30 degrees and 24 degrees, induction for longer time points (upto 24 hours), different IPTG concentrations at different induction temperatures (0.1 mM, 0.5 mM, 1.0 mM). I get good GST expression even with 0.1 mM IPTG, at 30 and 24 degrees respectively. I plan to use 20 degrees and lower temperatures for induction next. Can I use BL21DE3 for these tempeartures? I have also read about using special bacteria, Arctic express for lower tempearture inductions. Has anyone tried using this train. If anyone has any suggestions, I would be really appreciate it.

Thanks.

Priya

Silly question, but have you checked the codon bias etc?

Next, do you have any suggestion that the protein might be toxic to your cells? If so, and if you are growing cells to saturation overnight before the actual induction, you might have leaky induction and you might be selecting against your protein-containing cells...

The reason I say all this is that I've just heard William Studier, of pET expression fame, give a brilliant talk at a protein expression meeting. He put out a paper in 2005 in Protein Expression and Purification Vol 41: 207-234. If you want a pile of tips from a giant in the field, check it out!

Here's a few I heard: addition of Mg to 2 mM can increase the max OD from 3 to 18.

100 mM phosphate (for buffering) and amino acids inhibits kanamycin.

Tryptone from casein can have trace levels of lactose, which can cause unintended induction (this is the cause of the leaky expression that kills off your good clones).

If you use his fully defined media, you could get a final overnight OD of not 4, not 14, but 40. Imagine how much protein would be in that many cells!

[Thank you for the suggestions. I am going to read that article you suggested. Hopefully it will clear up some of my doubts.

I wanted to mention one another issue. I have my protein in pGEX vector. I have been reading that BL21 DE3plysS cells are good basically for T7 promoters. I still get GST protein expression under these conditions though. Any thoughts?