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Protein folding in different species


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#1 2xzwei

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Posted 17 May 2013 - 06:36 AM

Hi!

I've overexpressed a protein in both bacteria and human cells (293T). This protein is immunomodulatory and can inhibit TNFalpha secretion of immune cells. So I designed a wildtype and also mutated form that should be expressed by both species.

Now I have this curious findings:
The WT protein from both bacteria and 293T is working well in-vitro (positve effect).
The MUT protein from 293T cells has no effect at all (that's alright as it should serve as negative control).

BUT: The MUT protein from bacteria is also able to abrogate TNFalpha secretion (which should not be the case!).

I've confirmed the sequences of the vectors that have been used for transformation and they were okay. Also the isolated plasmid from bacteria carries the mutation and is fine.
Furthermore the MUT protein from 293T is recognized by ELISA, but NOT the MUT protein from bacteria (although they should be the same). So I'm wondering whether the folding of the MUT protein is different in human 293T and bacterial cells?! Or what explanation would you give for this fact?

I'd be grateful for some advice...
Regards :)

#2 DRT

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Posted 19 May 2013 - 11:42 AM

Hi!

I've overexpressed a protein in both bacteria and human cells (293T). This protein is immunomodulatory and can inhibit TNFalpha secretion of immune cells. So I designed a wildtype and also mutated form that should be expressed by both species.

Now I have this curious findings:
The WT protein from both bacteria and 293T is working well in-vitro (positve effect).
The MUT protein from 293T cells has no effect at all (that's alright as it should serve as negative control).

BUT: The MUT protein from bacteria is also able to abrogate TNFalpha secretion (which should not be the case!).

I've confirmed the sequences of the vectors that have been used for transformation and they were okay. Also the isolated plasmid from bacteria carries the mutation and is fine.
Furthermore the MUT protein from 293T is recognized by ELISA, but NOT the MUT protein from bacteria (although they should be the same). So I'm wondering whether the folding of the MUT protein is different in human 293T and bacterial cells?! Or what explanation would you give for this fact?

I'd be grateful for some advice...
Regards Posted Image


addition of functional groups?




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