Problem with recombinant protein expression

[soundlover]

hello all,

a friend of mine has built a plasmid to express a recombinant protein in yeast, but she´s having problems in expressing the protein, the level of expressed protein being to low. i do not recall what is the vector used, the promoter is Gal4.

after that result, she re-analyzed the sequence and discovered that immediately before the ATG of the insert (~10 base pairs before], there is another coding region (~50 bp) that is in frame, and is encoding a small peptide.

something like this:GAL4------ATG--non desired protein-TAG---10bp--ATG-INSERT-TAGthe question now is: it is possible that the transcription of the small peptide influence the transcription of the protein of interest?and the distance between the two coding regions, does it matters?

tks in advance

 

[soundlover]

probably this should be in the chemistry section. my bad

 

[Monique]

I don't have much experience with recombinant proteins, but did she check whether the short form is actually expressed? (isolate cDNA).

 

[soundlover]

I don't have much experience with recombinant proteins, but did she check whether the short form is actually expressed? (isolate cDNA).

no

i updated the initial post with:

and the distance between the two coding regions, does it matters?

 

[iansmith]

Having more details about the expresssion vector and the origin of the protein could help.

 

[Charion]

As mentioned, more info is needed to give a definite answer. In principle it is possible that the smaller peptide gets transcribed preferentially. Also you were talking about low yield.
Is the measured protein really the one in question? That is, have you analyzed the protein sequence (e.g. with MS/MS)?
On a different note, there are gazillions of possibilities why overexpression of proteins fail, including the fact that it may disrupt normal cellular function.

 

[Ygggdrasil]

In eukaryotes, the ribosome generally scans from the 5' end of the messenger RNA to find the first start codon (ATG) and initiates translation from there (in contrast, prokaryotic translation initiation is directed by specific sequences on the mRNA). So, the first ORF is likely being translated and the gene of interest is not.

 

[Charion]

Hmm I am not sure what you mean that prokaryotic translation is initiated by specific sequences. Do you mean Shine-Delgarno sequences?
To clarify, maybe. The main difference is that prokaryotic genes are often organized in operons, resulting in transcripts that possesses several genes, often (but not always) carrying individual Shine-Delgarno sequencces.
Eukaryotes generally do not possesses operons (though yeast is a little special in this regard). The transcriptional start is defined upstream of the aug by the Kozak sequence. So, in case there is a complete transcript, which includes both ORF (can easily tested by RT-PCR), there will only one Kosak sequenece, so chances are that the first ORF will be translated preferentially. But as the sequence is so short, there is a fair chance that the second will also be translated. That is why I was asking how the measured protein looks like. Also the question is where the tag (if any) for the protein purification is located.