Dear
colleagues,
Thanks
for the many approaches to address this problem. I neglected
to mention that
1. We
have an N-terminal His6 tag to the MBP-fusion and first
purify the complex with Ni-NTA. Thus, it’s likely that
either the fusion protein is getting cleaved or it’s a
transcription/translation problem.
2. We
get approx. 50:50 MBP and fusion but the total MBP +
MBP-fusion are very low.
3. We
can partially separate the two by size exclusion.
4. We’ve
not had much luck with other tags, such as SUMO and GST.
Best
regards,
Elias
Dear Elias,
While, as suggested by Mark and others, there can be many
reasons for your problems, such as your fusion protein being
largely insoluble, I would second Michael's suggestion to put
a His tag in front of the MBP. Ni-NTA has much much higher
capacity than amylose (and is also much cheaper), so you can
increase your yield hugely by using the MBP merely as a
solubility enhancer, combined with a His tag as the actual
affinity tag. It has the additional advantage of getting rid
of host MBP of course.
I don't know which plasmid you're using, or if you secrete
your fusion protein or not, but an easy way to put a His tag
in front of the MBP that I used once was to clone MFG (my
favourite gene) into pMAL-c2x (i.e. the cytosolic version),
and then cut out the whole MBP-MFG construct from the plasmid
with NdeI (cuts just 5' of malE) and whatever I had used at
the 3' end of MFG, and put that into pET28 - thus generating a
His6-(thrombin site)-MBP-(factor Xa site)-MFG fusion. I had
had problems with low yield with the original pMAL-c2x
construct, but this His6-MBP construct solved all my problems.
Cheers,
Julia
On 15/02/16 15:39, R. Michael Garavito
wrote:
Elias,
Your experience is not unusual, in fact
it is completely expected unless you work with a MalE null
mutant, which I don’t think you can get versions of the
usual BL21 expression hosts. We always want to see native
MBP as it means the amylose column is working (see note
about amylase destruction of the amylose resin) .
However, I can see that you don’t want to use 20-30 mL of
expensive resin just to ensure that your MBP fusion
protein is not out competed by the native MBP for the
amylose resin. If you have a real gene jockey at hand,
you might engineer a N-terminal fusion to the MBP so that
you create a His6-MBP fusion protein. Keep the normal
start/signal peptidase cleavage site, but add a 6- or
10-His tag in front. Run a single step Ni-column to
remove background protein and the native MBP, the load the
elution fraction on an amylose column.
If you are not secreting your MBP
fusion protein, another option is to pretreat your cells.
Native MBP is in the periplasm (at concentrations up to 1
mM). There are protocols to pop open the periplasm (using
high [Ca2+] or [EDTA]) then centrifuging your cells; the
native MBP remains in the supernatant. People have used
this method to replace native MBP with labeled MBP to
study periplasmic function (see work in the 1980s by
Nikaido’s group). Most of the time the cells are not
broken, but I have never tested this method on a large
scale (4-20 wet cell wt.).
I also noted that you are not using the
typically recommended host from NEB with the REP3 plasmid.
While it may not be an issue, the NEB host has reduced
amylase production which can chew up your amylose resin
faster.
****************************************************************
R.
Michael Garavito, Ph.D.
Professor
of Biochemistry & Molecular Biology
603
Wilson Rd., Rm. 513
Michigan
State University
East
Lansing, MI 48824-1319
Office: (517)
355-9724 Lab: (517)
353-9125
FAX: (517)
353-9334 Email: [log in to unmask]
****************************************************************
Dear
Colleagues,
We are attempting
to express our target protein fused to MBP in
BL21(DE3) and BL21(RIPL). Unfortunately, we get
a mixture of MBP-target protein and MBP only,
which severely depletes our overall yield of
MBP-target protein. We've tried enriched media,
such as 2xYT & TB and also optimal
high-density induction in minimal media at
temperatures ranging from 20-37deg and IPTG from
0.05-0.5microM.
Any suggestions to
address this competitively high yield of
MBP-only protein?
Cheers,
Elias
--
Dr. Julia Griese
Postdoctoral Researcher
Stockholm Center for Biomembrane Research
Department of Biochemistry and Biophysics
Stockholm University
106 91 Stockholm
Sweden
phone: +46-(0)8-163 246
email: [log in to unmask]