Expression of protein

[shifali chatrath]

Hi all!
I also have expression problem. I tried expressing my protein, cDNA cloned in pET19b in Rosetta gami cells ( beacause it has 5 disulphide bonds and cDNA is from snake)and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation)
Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from expression hosts. DNA is there but no protein.
Any comments??????? 

On Sat, 04 Apr 2009 DK wrote :
>In article <mailman.735.1238791630.13724.methods from net.bio.net>, Basia Dymek <bb-bb from gazeta.pl> wrote:
> >Hi!
> >I have prepared a plasmid using pFLAG-CMV-2 vector (Sigma) into which I have
> >cloned my gene of interest. Finally, I have some level of expression after
> >transfection HepG2 cells, however it is unsatisfactory for me.
>
>Unfortunately, when it comes to overexpression in mammalian cells,
>the typical protein levels are unsatisfactory to most...
>
> >CMV promoter
> >should be the strong one. Have you got any clues what could be the reason of
> >low level of expression of my protein? I would be thankful for any advice.
>
>The vector is for transient expression. Thus, if your transfection is
>inefficient, expression level will be low no matter what. Make sure
>you transfect at least 50% of cells. Lots of things involved, starting
>with s high quality plasmid prep and ending with the best transfection
>method. Beyond that:
>
>Besides the strenth of promoter, many factors affect expression levels:
>rate of transcription is influenced by the gene's sequence, mRNA stability
>varies, rate of translation may be influenced by mRNA sequence/structure,
>and protein stability varies quite a bit. In addition, overexpression of some
>proteins is toxic to cells and inhibits transcription/translation machinery.
>Most of these you can't change rationally, so just accept a fact of life that
>some proteins express at higher levels than others.
>
>DK
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449