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

You should read a very good article

Escherichia coli Host Strains
by Nicola Casali
in: Methods in molecular biology, vol.235: E.coli plasmid vectors, Edited by N.Casali and A.Preston Humana Press Inc., Totowa, NJ

And another one:


Strategie for Achieving HIgh-level Expression of Genes in E.coli
S. Makrides
Microbiological Reviews, Sept 1996, p 512-538

And another:

Genetic design for facilitated production and recovery of recombinant proteins in E.coli
Jonasson P et al, Biotechnol. Appl. Biochem. 2002, 35, 91-105

hi chempilot,
if you mean the pET vectors from novagen it's very interesting because one of invitrogens people, from which i get the pET101-vector i used in my fallen experiments (and likewise pratik use) told me, that novagen got the license for this system
so the question is why this system runs and ivitrogens not...

[quote name='pratik' date='Oct 5 2005, 11:05 PM' post='26838']
[
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
[/quote]

hi pratik,
unfortunately no expression
last week i got an new kit from invitrogen but at this time i can't say if it works better, maby next week i have new results. at least i can see an increasingly protein band after induction with IPTG with the new kit. from uninduced to induction for at least 4 hours the band seems stronger. but not like it should be i think. but unfortunately just for one protein, the other ones seems toxic for the e. coli strain. i think, i will test another strain like alesia adviced

Hi,
Thanks for your suggestions Alesai, pesji, chempilot , flausch..

I tried to optimize the overexpression of my protein in the BL21 DE3 cells hoping to out compete the contaminating 20kd band binding on the NI column.

This time I induced my cells for six hours using 2Mm , 4Mm , 6Mm and 8Mm IPTG , i harvested 1 ml of the culture , spun it down, lysed the cell pellet completely with 80ul of SDS PAGE sample buffer, boiled it for 5 minutes at 100, spun the boiled sample down at 13000rpm for 5 minutes and loaded 15ul of the supernatent on a 10-16% tris glysine gel.

This time i did not see a doublet[but I wasnt quite sure of it] at 37KD where my protein should be but a single thick band in all 4 concentrations and of course the 20KD non specific protein at the bottom of each lane.[See the attached gel picture].

I confirmed the identity of my protein using a monoclonal anti HIS antibody and the thick band at 37KD where my protein should be lit up indicatig that I am able to overexpress it slightly, AND THE 20KD BAND ALSO LIT UP.

I am confused why did I not get a doublet this time?

why do i see large amount of protein in my whole cell lysate but not the supernatent that i load on the NI column which yields the DOUBLET?

does that mean my some of my potein is ending up in the insoluble fraction?

Based on my gel picture is this amount of expression enough for large scale purification?

And finally how does the nonspecific 20 KD protein from BL21 binds to my monoclonal???

Attached is a gel picture showing induction of my protein: the one at the second band after the first pink band on the ladder [37 KD]

hi pratik,

thanks for showing us your gel photo! I can still see a additional band below your 37kDa protein, but I'm not sure.
I think you missed one thing there: control lane which contains protein expression profile before induction. Next you need to figure out if this 20 kDa comes from e.coli's own proteom or it is truncated product of your fusion protein.

Another simple experiment you can do: using Ni colunm to "purify" whatever in E.coli cell BEFORE IPTG induction. You can see whether the "doublet" or "20Kda" still show up or not.

The above-mentioned test will further give you some hints of what exactly happened during your purification process.

Good Luck!

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