Hi Ganesh,
two things I'd advise that might make a difference for you:
1. Improved media for better expression
See "Protein production by auto-induction in high-density shaking
cultures" by W.F. Studier Protein Expression and Purification 41 (2005)
207-234
(there's also a recipes paper that you'll need to dig out)
I've found that an LB based complex autoinduction media comprising of
LB supplemented with the 50xm buffer mix, 50x 5052 carbon source mix, 1000x
metals, and MgS04 really boosts expression levels every time (recipes are in
the Studier papers). If you don't want to use autoinduction, just leave out
the lactose from the 5052 carbon source mix and induce with IPTG as normal.
You should get a higher OD and probably higher protein per cell.
2. Removing periplasmic materials prior to lysis might improve His-tagged
purification yields
I saw this recently and am about to try it, but haven't yet. Anyway, it
may be useful in your case. The principle is that you use osmotic shock
to take out materials in the periplasm that bind / strip Ni columns and
reduce IMAC yields. After the shock step you lyse and purify normally,
and in theory get better retention of your protein with less contamination.
Full details in:
"Enabling IMAC purification of low abundance recombinant proteins from
E. coli lysates" nature methods | VOL.6 NO.7 | JULY 2009 | 477
There is also a supplement to this on the nature methods website, which
you will need.
By combining the above you ought to obtain more of your protein to start with,
and then capture more of it on the IMAC first step, and hopefully with greater
initial purity. You should then be better able to clean it up further if need
be and still have something left to work with at the end.
Good luck,
John.
john.hi...@syngenta.com
www.syngenta.com
-----Original Message-----
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Phoebe
Rice
Sent: 26 August 2010 20:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Problems in purification
Have you tried expression tricks like Rosetta cells? Testing different
colonies and/or starting from fresh transformants? Sometimes that matters.
If your protein is an oligomer and your contaminants are degradation products,
you might try adding some urea. If desparate, you could spike the fraction
collector tubes with EDTA when you run the Ni column, as well as using the
usual protease inhibitors.
Phoebe
=====================================
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.rsc.org/shop/books/2008/9780854042722.asp
---- Original message ----
>Date: Thu, 26 Aug 2010 12:07:45 -0400
>From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Matthew
>Bratkowski <mab...@cornell.edu>)
>Subject: Re: [ccp4bb] Problems in purification
>To: CCP4BB@JISCMAIL.AC.UK
>
> Hi.
> What size are the impurities? If they are smaller
> than your protein, then they could actually be
> truncation products, which will be difficult to
> purify away since they maintain some of the same
> characteristics as the full length protein. You
> can check for C-terminal truncations using a
> His-antibody, but N-terminal ones will be harder to
> detect. If the impurities are larger (particularly
> if they are around 70 kDa), you could be looking at
> E. coli chaperones.
> To improve, the purity of the first Ni-NTA step, I
> would include a more stringent wash. How many
> column volumes do you wash with now, and how high of
> imidazole concentration? You can go up to 20 mM
> Imidazole in your wash. You could also include
> some glycerol in your buffer (up to 10%) and
> betamercaptoethanol (around 5 mM) to break
> non-specific protein interactions. For ion
> exchange, run a shallow gradient and include more
> column volumes of wash before elution. For the
> third step purification, I would recommend using
> size exclusion chromatography. Either Superdex 200
> or Sephacryl S-100 would probably work to remove
> some impurities as long as the impurities are a
> different size than your protein of interest. I
> would use between 150 mM - 1 M NaCl in the buffer,
> depending on how strong the non-specific interaction
> is, and 1 - 2 mM DTT. Make sure to collect small
> fractions (0.3 - 1.5 mL) to reduce contamination
> from nearby peaks.
> Matt
>
> On Thu, Aug 26, 2010 at 8:24 AM, ganesh pathare
> <ganeshpath...@gmail.com> wrote:
>
> Dear all,
>
> I have problems in purifying a protein. The
> protein is 38,000 daltons and has a N-ter
> His-Tag. The protein expression levels are low
> and as a result I have a limit for the
> purification steps.
> Initially I used NiNTA columns with 50 mM sodium
> phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM
> Immidazole for the affinity purification, but it
> contains lot of impurities. I varied the salt
> concentrations out of which I could get optimal
> results at 20 mM NaCl concentration but still the
> amount of impurities was more.
> After affinity purifications I used Ion exchange
> chromatography using MonoQ column (25 mM tris pH
> 7.5, NaCl 0 to 1M) which could not seperate the
> protein from the impurities. I also tried using
> Hydrophobic interaction chromatography (Resource
> Ether, Phenyl sepharose, Resource
> Isopropyl) instead of ionexchange chromatography,
> which resulted in better purification of the
> protein, but the problem is I get very less
> protein after this step and there are still two
> major impurities. The buffer conditions for HIC
> was (1.5 M ammonium sulphate, 25 mM Phosphate
> buffer pH 7).
>
>
> I would be very greatful if someone could help me
> in this concern.
> Thanks in advance.
>
> Regards,
> Ganesh
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