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Weird Induction Problem - (Jan/15/2010 )

Hi everybody,
I m trying to induce my protein (ca 25kDa) with 0.6mM IPTG for 2h in BL21(DE3) cells @37C. The construct is pET11d and my protein is having no tag. The problem is the overexpression of unknown,weird protein around 20kDa. I dont understand why this happens and since the molecular weight is very similar and the expression of the protein of interest is much lower than this weird protein, I can not get rid of this protein during further purification steps.

Does anyone have any idea what is going on and how to fix this problem??
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-1324409-

1324409 on Jan 15 2010, 05:51 PM said:

Hi everybody,
I m trying to induce my protein (ca 25kDa) with 0.6mM IPTG for 2h in BL21(DE3) cells @37C. The construct is pET11d and my protein is having no tag. The problem is the overexpression of unknown,weird protein around 20kDa. I dont understand why this happens and since the molecular weight is very similar and the expression of the protein of interest is much lower than this weird protein, I can not get rid of this protein during further purification steps.

Does anyone have any idea what is going on and how to fix this problem??


I consider that your pcr product and designing is perfect, and you have sequenced the plasmid and confirmed the identity of your insert.

I would suggest you start off with 0.7 od culture @ 37 degrees, induce with 0.1-0.2 mM IPTG @ 25 degrees for upto 4 hours, take samples every hour and analyze them on SDS PAGE.

If this shows the same things. Perhaps, you should do a western blott if you have happen to have an antibody perferably at C term and/or N term, which would confirm if your protein is getting truncated prematurely or not..?

Hope this helps.

Let me know what happens...

Cheers,

Kaushik

-KAUSHIK THAKKAR-

If the "weird" protein has the same chromatographic behavior as your protein of interest, the weird one could be a truncation of the one you want----ie, the translation of the weird protein begins at a downstream ATG of the overexpressed RNA.

How to solve? I think as said above, you can try overexpressing at different temperatures to see if this helps. Another possibility is to use a BL21 (DE3) strain that has extra tRNA's for rarely used codons. I have seen protocols that use ammonium sulfate cuts to separate a truncated from from the full length. Good luck.

-StevieRay-

Thank u guys for the replies.
Yes, i sequenced my plasmid and my insert is having no obvious problem that took my attention but I ll double check it. I did a site directed mutagenesis to construct my plasmid. Perhaps stg has changed during the procedure about the backbone, which is of course not sequenced (only insert was sequenced) By the way,I did a Western to check the induction and I can hardly see my protein induced and the weird protein (fat band visible on Ponceau) was not detected with the antibody for my protein. But since the Ab I m using is monoclonal, I can not exclude the possibility that the weird protein is a truncation of my protein so far. Epitope that the Ab recognizes might have been lost with the truncation. I will probe it with another polyclonal Ab to be sure about the identity of the weird protein.

We also thought of making transformation with the empty vector and do the induction to see whether the vector backbone is ok or not. If there is also no problem with the backbone, I try different induction conditions like u suggested, differing temp. and so on. Other than that nothing came to our minds so far. Let me see how it goes. I ll keep u updated <_<)

Cheers,
Volkan

-1324409-

You might just be seeing the T7 RNA polymerase. The pET manual says that it is ~24 kDa.

-swanny-

Hi guys,
The problem is solved, it was the bacteria which was somehow contaminated. T7 Poly. by the way is around 100kDa i guess. It can not be 24kDa if i m not mistaken.

-1324409-