You've tried the obvious things. Lowering the IPTG concentration doesn't
really work all that well, at least in my hands. We find we can still
get maximal expression from many promoters like trc with only 0.2 mM
IPTG, and the response doesn't modulate well: it's either on or off.
Growing at lower temperature, like 18-22 deg C sometimes works, but
sometimes not. Here are a few more approaches:
1. Use "leaky" expression in a pET vector system. Just don't induce it
at all and grow for 4-18 hours. The slow expression may allow for
protein folding before kinetic trapping in protein tangles. You may
want to combine this with lower temperatures and less rich media
(e.g., LB instead of TB) to slow growth and protein synthesis.
2. Put multiple, tandem copies of your gene behind the promoter. I'm
not sure how this works, but it may tie up enzymes transcribing the
message from the plasmid to slow mRNA transcription and thus
translation. I never really understood the chemistry behind this,
and no one could explain it to me in a cohereht way. However it
works, it can slow protein expression enough to allow for soluble,
instead of precipitated protein. This works like a charm to express
the alpha subunit of trp synthase. The vector has 4 tandem copies of
the gene after a trc promoter. You can make 1000 mg of soluble
protein per 100 mL of culture (!) (I didn't mess up my zeros.)
3. Express your protein with a solubilizing tag on the N-terminus. I
think there are some commercial vectors that put NusA in front of
your gene. This may allow for soluble expression. NusA is supposedly
better than 6X His tagging for making soluble protein. (Hasn't
worked for me yet, but the literature suggests some successes.)
4. Try a different promoter. We use a modified version of pTrc99 that
has at trc promoter instead of the T7 promoter. It is tightly
controlled by IPTG, with no leaky expression. It can be a fierce
promoter, but it may not be as fierce as T7, and can give you
different results.
Have fun! And good luck!
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [email protected]
On 10/17/2014 8:51 AM, xaravich ivan wrote:
Dear cc4bb enthusiasts,
This is slightly off topic but many protein crystallographers might be
familiar with this problem.
I have been trying to over-express a bacterial (non-E.Coli) protein
in E.Coli and more than 80% goest to inclusion bodies.
I tried the following
Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM -
0.5 mM)
Cold shock for 30 minutes in ice before induction
Slow rotation speed at 18 degrees O/N after induction
While these steps helped a bit I still get about 50-60% of my protein
in inclusion bodies.
I would like to know what other steps do you suggest to enhance the
yield in the soluble fraction (without changing the host strain or
manipulating the DNA)
Thanks in advance
Ivan
