8 replies to this topic

[Missle]

I've been working with a highly soluble protein (predicted to be by 3 different prediction softwares & experimentally confirmed) yet am having trouble with low expression in e.coli - BL21(DE3) & Rosetta 2 (DE3).  Have tried several different media formulations, IPTG amounts (0.1mM to 1.0mM), and expression times (2hr to 22hr) and have not been able to significantly boost expression.
Most recently, have been expressing overnight @ 25C and inducing w/0.1mM IPTG.  The OD600 has increased to up to 10 but my protein levels have not increased.  Does anyone have an idea why the protein expression would not increase?  Any tricks for boosting expression (I'm in the 100 - 300ug/L range)?

Thanks, Thanks, Thanks!!!

[Papaver]

I guess you've already checked the plasmid if it is correct? How do you check the expression level? Is there a significant difference between before and after induction?
I once had to add glucose to the media (I think it was 0.5 %) which increased the expression...I was really suprised about the effect.
Maybe you can give some more information. Was the protein expressed by other persons so you can repeat their protocol?

[Missle]

Thank you for taking the time to respond.  I have sequenced my insert and it is correct & in-frame.  I've been checking relative expression levels by SDS-PAGE and I've purified a few 1L productions to know that my yield is low.  I can see an induced band when comparing pre- and post-induction but it is not really strong and it is barely visible when looking at the Total Cell Protein samples.  Western blot & ELISA work has confirmed the material I purified is my target protein.
My plasmid has ampicillin resistance so I've been using carbenicillin and have tried adding glucose (1%) to the media but so far no improvement.  Do you think I could be adding too much glucose?  Most recently, I did an overnight expression in terrific broth, which has 0.4% glycerol.
There are some references in the literature but they are vague on protocol (BL21(DE3) cells, induce with 1mM IPTG for 3hrs).
I've also expressed at 37°C for 4 hrs and seen some expression (this is the condition I've run my 1L expressions) but nearly what I think I should be getting.....

[Papaver]

Up to 1 % glucose is fine...
I guess you are using pET-Vectors for expression...and just to make sure; BL21(DE3) does not contain an additional pLys-Vector, does it? When I had once the problem with expression it was a "host" problem and I could manage it with adding glucose. Then I reduced IPTG concentration to 0.5 mM and induced overnight at 16 °C. I also could enhance the yield by adding IPTG at a OD600 = 0.8
Have you ever checked if your plasmid is still there at the end of induction (or in between). Might your protein be toxic? Some are also adding argenine to the medium to help protein folding. Does your protein maybe degrade?

We don't really know why but in our lab we cannot express a protein tagged N-terminally with a strep-tag (IBA-pASK-system)...you can see an expression in 25 ml cultures but there is almost nothing in 1 L culture. A colleaque of mine is expressing her protein in many 250 ml cultures as a result which is pretty annoying but there is no other way. In contrast when using the same plasmid system with an c-terminal strep-tag there was a good expression

Maybe, if nothing helps you should consider another expression system...

Edited by Papaver, 16 June 2012 - 09:39 AM.

[Missle]

The host cell line is not a pLys.  I haven't tried inducing at below 25°C but perhaps that is where I should go next.  I have tried inducing at OD600 = 0.8-1.0 instead of 0.5.  I now have a construct where the His tag is on the C-term and will try this to see if I get better results.
I don't believe my protein is toxic but how do you determine this?

[Papaver]

Missle, on 18 June 2012 - 09:22 AM, said:

I don't believe my protein is toxic but how do you determine this?

Literature research...what is the natural function of the protein...

Good luck for your further experiments...

[Missle]

Quick update - I've done an overnight expression (25°C) with or without glucose in the media and purified plasmid pre and post-induction.  I'm not loosing my plasmid.  There is also nothing to indicate that it would be a toxic protein in the literature.  Still researching more options.....

[Papaver]

Might codon usage be an issue (is it a bacterial protein)? BL21(DE3)-CodonPlus-pRIL can be used therefore.

Missle, on 15 June 2012 - 11:46 AM, said:

I've been working with a highly soluble protein (predicted to be by 3 different prediction softwares & experimentally confirmed)

How was the stability of your protein experimentally confirmed?

[Missle]

Problem solved!  The problem appears to have been prophage instability of the (DE3) cell strains.  I switched to BLR (DE3), which aids in plasmid and prophage instability, and have robust soluble expression.