Questions about gene expression, codons, and Kozak sequence - (Jan/10/2008 )
How is every body?
I'm trying to find the answers for the following questions and I need your help please since this is the firs time for me to work with expression vectors and transformation.
the questions are:
1. What to do to confirm the presence of gene of interest in the expression vector? (i know that we can do sequencing, but is there any other way?)
2. What to do if the gene of interest is not cloned in frame with N-terminal V5 epitope? and how can we detect this problem?
3. Should the gene of interest contain a stop codon? Why?
4. Should the gene of interest contain a Kozak sequence and an ATG initiation codon? Why?
5. Can we use expression vector to allow the expression of C-terminal V5 epitope with ORF clone? Why?
I'm still searching for the answeres because I'm using ORF clone as my entry vector with an N-terminal V5 epitope fused with the inserted gene and i need to do transformation and then transfection of vero cell line to express the inserted gene.
So, I will really appreciate your help
Hi there, and Welcome to Bioforums!
I'll try to anwer your questions to my best knowledge.
1. There are many ways of testing the presence of your gene in the vector. You may try to digest your plasmid with restriction enzymes before and after your insert and check the the size of the products on gel. You could desing primers and run a PCR on your product, etc.
2. You have to figure it out before cloning.
3. Yes. In order to stop the translation, the cell machinery needs a stop codon.
4. The Kozak sequence facilitates the translation of the cDNA by the ribosomes. But since you have an epitope at the N terminal, adding it the your gene would not help the expression. Kozac sequece have to be placed before the first ATG, witch in your case is in the V5 epitope. As for the ATG in your gene, its presence it not required, since the translation will begin to the V5 epitope. But since its presence may be required for your protein'S function, you should let it threre.
5. Never heard of these vectors, but why not! Just make sure you put a stop codon after the V5 epitope and remove the one on your gene. Keep in mind the frame!
I have heard somewhere that some tags have less negative effects on one or the other end of the protein. You may want to check this with the V5 epitope.
Hope this helps!
2. If there is an error in the plasmid and the gene is not in frame with the epitope, you can confirm it by sequencing.
5. You can allow expression of epitope in C-terminus or N-terminus as long as they are in frame. usually people use epitope tags to detect them as there is commercial antibody for every single protein available. Proteins could behave differently because of the attached epitope tags, it could be either C- or N terminus or even both.
4 - normally your vector contains the kozack sequence and an appripriate ATG before coding sequence for V5 epitope. You can left the atg of your gene of interest but start directly with the open reading frame of the gene of interest.
5- if you can appropriately clone your ORF at 5' of the V5epitope coding sequence, yes you can express the gene of interest
Thanks alot for all of you
this is really helpful
thank you very much
and nice to meet you