mRNA secondary structure and protein expression level

Trond Erik Vee Aune trondaun at chembio.REMOVETHISBEFOREREPLYING.ntnu.no
Wed Sep 11 01:47:29 EST 2002


Dear Daniela,

I am currently studying mRNA folding related to expression levels. What 
we have seen is that MFOLD will often predict secondary structures that 
covers SD or start ATG, but the expression levels can be fine. It is 
also said that the individual bond should be of at least -6 deltaG to be 
of any relevance, that is, to hinder translation, or translation 
initiation (Maarten H. de Smit and Jan van Duin, 1994).

In your case it seems you get no protein at all so I don't think 
secondary structure is the problem here. Btw, hos sensitive is your assay?

You also mention codon usage. What organism do the gene come from, and 
in what organism do you try to express it? I've expressed different 
human proteins and different bacterial proteins in E.coli without any 
codon optimization, it has been of no problem.

One more thing, if you try to express a protein that needs disulfid 
bridges to obtain right confirmation, and you use ELISA assay to detect 
your protein, then missing disulfid bridges can be a problem. Antibodies 
may not bind to insufficiently folded proteins, so in your case it can 
be that the gene is expressed but because of misfolded protein it is not 
detected. This is mostly a problem when trying to express proteins in 
E.coli and not using a signal peptid (the protein is not being 
translocated to periplasma).

Trond Erik





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