Appearence of non specific[non 6His tagged] proteins with Ni-NTA purification. - (Oct/04/2005 )

Hi,
I have cloned a gene [Polyamine binding Protein] from a gram positive bacteria into pET101-D-TOPO vector from Invitrogen.I have checked my sequence and the gene is intact. I am using BL21 DE3 cells for expression.

I did a pilot expression profile when I induced my protein with 1Mm/0.7Mm IPTG for 6 hours and performed a Western Blot with a monoclonal anti His antibody and a single discrete band [38kd]-which is the size of my protein lit up from 1-6 hours with increasing intensity, but on the SDS Page gel my protein was not the most dominant band and by no means looked like it was over expressed.

I tried to purify my protein by using Pierce B-PER [Cat. No 78100] NI-NTA pre-packed purification column and buffers.

In my elution’s a 25KD protein [A thick band], A Faint doublet [At the expected size of my protein] and a band at 42 KD showed up.
But the 25kd protein [A thick band] was visible in my flow through with all my washes with wash buffers.

I performed a western blot with my elutions and again a single faint band lit up, apparently one of the doublet bands is my protein that is 6His tagged.

I increased my Imidazole concentration to 40Mm in my first wash buffer, No Imidazole in wash buffer 2 and 200Mn Imidazole in wash buffer 3[Pro-ex Buffers compositions]

After loading a 200 ml lysate with a 40 Mm wash lots of proteins showed up in the flow through, very few in the wash with buffer 2 and with 200Mm a 25kd protein[A thick band], A Faint doublet[At the expected size of my protein] and a band at 42 Kd showed up-I.E the pattern was unchanged.

How do I get rid of these non specific background bands?

Can anyone help me with this??

e. coli is known to have many histidine rich proteins that will also bind to the 6His column. Also, there are many IPTG inducible protiens in e. coli. so, your sample is never going to be 100% pure. most of the Ni-6His literature claim ~95% pure, which is about right. it sounds like your protein is there, but you will probably have to use another purification method, such as ion exchange or size exclusion, if you want to get >95% purity.

hope that helps!

hi pratik,
unfortunately i have no idea for solving your problem because i'm using the same system and i have the same problems... therefore i'm very interesting in the pieces of advices given here in the forum. i'm working with two different proteins but unfortunately one of these proteins is about 20-25 kDa so it's very confusing. but good luck to you!!! i will checked the forum for new ideas

Try other strain. I can say a lot of good of TG1 strain; Also gamme of Rosetta strain helped me.

Also change the induction (promoter). I have several proteins under tet promoter, i induce with anhydrotetracycline, and have no problems.

With IPTG I also saw very often a big band of one E.coli chaperonin Gro EL (correct me, if I do not remember exatly its name). This chaperonin adore NI-NTA column

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hi pratik,
unfortunately i have no idea for solving your problem because i'm using the same system and i have the same problems... therefore i'm very interesting in the pieces of advices given here in the forum. i'm working with two different proteins but unfortunately one of these proteins is about 20-25 kDa so it's very confusing. but good luck to you!!! i will checked the forum for new ideas
[/quote]
Hi Flausch,
Sorry to hear about that, by the way have you been able to overexpress any of these proteins? , I cant seem to get my protein overexpressed.
Pratik

the ion exchange columns from Amersham are cheap and work very well. i have been using the HiTrap Q HP column for a while with great results.

http://www4.amershambiosciences.com/aptrix...ent/na_homepage


Alesia's right. if you have used that much IPTG and saw no change, a new promoter/vector/E.coli strain may help. i had a problem with Gateway vectors expressing and once i changed to pET vectors i got tons of protein.

Is that possible that contaminant protein forms disulfide bond with your 6His fusion protein?
Could you try to add some more beta-mercaptoethanol?

Good luck!

I had express now several recombinant proteins with pET blue vector and mostly Tuner pLAC cells and I have seen this contaminant from time to time. It's usually expressed in a differential manner.

If your protein is weakly expressed (like it seems to be your case) then you get lots of the contaminant E.Coli His protein. One of our diploma student even deal for some times with a contaminant protein thinking it was the good one cause it had the same size

Trying different strains is a very good choice and also screening more clones even if the sequence is correct I would just pick three or four different clones and check. I had best results with Tuner pLAC and Tuner pLys (I'm dealing with either GC rich or AT rich sequences)

Some rec proteins are also binding poorly on HisTag resins so it might ends up in the wash as well, increasing imidazole in the wash neither during binding didn't help in most of my experiments !

Another possible trick described in a paper is to use anti his tag monoclonal antibody for purification of your protein but it also depend how your protein reacts to the purification buffer
here the link in Pubmed for this paper

pesji