4 replies to this topic

[Julio-Claudian]

Dear all,

I'm reading up on the types of expression system out there and I have a question regarding how an insert is expressed in a vector.

I have this gene of interest and putting the sequence into ORF Finder, I get a few possible reading frames (see attached image).



To cut to the chase, do (1) insert the gene as it is into the expression vector or (2) amplify the ORF and then insert it into the vector? Does it matter actually?

Upstream of the insertion site are the RE sites and protein Tags, would this change the way the insert is read when it comes to expression?

Comments please?

Thanks in advance.

[pDNA]

most of the time you know the protein sequence you are going to express and therefore know the corresponding ORF.

If tags are added they should not change the ORF they should just elongate the gene/protein N- or C- terminal. Its important that the first ATG after the ribosome binding site (in bacteria in a range within 7-13 nt) spans the right ORF with the protein sequence of interest.

It is not uncommon that a longer genes contains more ORF ...so you have to know what ORF you are looking for!

Regards,
p

[Julio-Claudian]

pDNA, on 24 October 2010 - 10:33 PM, said:

most of the time you know the protein sequence you are going to express and therefore know the corresponding ORF.

If tags are added they should not change the ORF they should just elongate the gene/protein N- or C- terminal. Its important that the first ATG after the ribosome binding site (in bacteria in a range within 7-13 nt) spans the right ORF with the protein sequence of interest.

It is not uncommon that a longer genes contains more ORF ...so you have to know what ORF you are looking for!

Regards,
p

Thank you pDNA. I believe that RBS are present in the expression vector (eg. pET).

Yes, I know the one I need and since I have the gene instead of the ORF (purple), I was wondering if it's ok that I use the gene and thus skipping another round PCR [for the ORF]. Then came your reply. I take it, from what you wrote:

1) that the start codon following the RBS should span the correct ORF; meaning I'd need the ORF (beginning at base 90) and NOT the gene I have now.

2) that a tag at the N- or C-terminus will not change the ORF

3) that RE sites at the 5' of the ORF will not change the ORF. Can I go on to state that the extra few amino acids before the fMET won't interfere with protein function?

[pDNA]

dreamchaser_jc, on 24 October 2010 - 11:10 PM, said:

pDNA, on 24 October 2010 - 10:33 PM, said:

most of the time you know the protein sequence you are going to express and therefore know the corresponding ORF.

If tags are added they should not change the ORF they should just elongate the gene/protein N- or C- terminal. Its important that the first ATG after the ribosome binding site (in bacteria in a range within 7-13 nt) spans the right ORF with the protein sequence of interest.

It is not uncommon that a longer genes contains more ORF ...so you have to know what ORF you are looking for!

Regards,
p

Thank you pDNA. I believe that RBS are present in the expression vector (eg. pET).

Yes, I know the one I need and since I have the gene instead of the ORF (purple), I was wondering if it's ok that I use the gene and thus skipping another round PCR [for the ORF]. Then came your reply. I take it, from what you wrote:

1) that the start codon following the RBS should span the correct ORF; meaning I'd need the ORF (beginning at base 90) and NOT the gene I have now.

2) that a tag at the N- or C-terminus will not change the ORF

3) that RE sites at the 5' of the ORF will not change the ORF. Can I go on to state that the extra few amino acids before the fMET won't interfere with protein function?

Concerning 1) ...so your gene does not start with a ATG? is it a prokaryotic gene? what is your definition of gene and ORF?

Concerning 3) ...they will change the strength of expression ...they do not code for amino acids sincte they lay before the ATG ...so they are in the 5' UTR. In pET vectors normally you go for the NdeI restriction site and add your gene of interest starting with the ATG.

Regards,
p

[Julio-Claudian]

pDNA, on 25 October 2010 - 12:54 AM, said:

dreamchaser_jc, on 24 October 2010 - 11:10 PM, said:

pDNA, on 24 October 2010 - 10:33 PM, said:

most of the time you know the protein sequence you are going to express and therefore know the corresponding ORF.

If tags are added they should not change the ORF they should just elongate the gene/protein N- or C- terminal. Its important that the first ATG after the ribosome binding site (in bacteria in a range within 7-13 nt) spans the right ORF with the protein sequence of interest.

It is not uncommon that a longer genes contains more ORF ...so you have to know what ORF you are looking for!

Regards,
p

Thank you pDNA. I believe that RBS are present in the expression vector (eg. pET).

Yes, I know the one I need and since I have the gene instead of the ORF (purple), I was wondering if it's ok that I use the gene and thus skipping another round PCR [for the ORF]. Then came your reply. I take it, from what you wrote:

1) that the start codon following the RBS should span the correct ORF; meaning I'd need the ORF (beginning at base 90) and NOT the gene I have now.

2) that a tag at the N- or C-terminus will not change the ORF

3) that RE sites at the 5' of the ORF will not change the ORF. Can I go on to state that the extra few amino acids before the fMET won't interfere with protein function?

Concerning 1) ...so your gene does not start with a ATG? is it a prokaryotic gene? what is your definition of gene and ORF?

Concerning 3) ...they will change the strength of expression ...they do not code for amino acids sincte they lay before the ATG ...so they are in the 5' UTR. In pET vectors normally you go for the NdeI restriction site and add your gene of interest starting with the ATG.

Regards,
p

The gene starts with an ATG, definitely, and so does the target ORF that I want. This all began with the realization that the amplified gene has a few possible reading frames and the longest of these (+3) began [with an ATG] at position 96.

At +1, the ATG at position one is only 114 bases before it terminates. So, herein lies the doubt which one to insert into the vector and hence our discussions.