In order to clone a gene (which is RT-PCR-amplified) into a plasmid and translated into its corresponding protein, Should the gene include a Kozak consensus sequence before the starting codon?
Thanks in advance
It depends on the plasmid and on the host. If you are planning to express the protein in a bacterial host no, you don't need the Kozak sequence. Just find an expression plasmid and examine the map/vendor information: there will be a "designated" start codon donwstream the promoter used for heterologous expression. You just have to insert the coding sequence in frame with that start codon.
Hope it helps!
The Kosak sequence is important to initiate the translation process in Eukaryotic cells. Therefore, you don't need to add it if you are going to express your protein in prokaryotic cells (e.g. bacteria). I always add the start codon (at the beginning) and the stop codon (at the end) to my interest gene sequence and works for me. Important note, you need to put the sequence of your protein in-frame regard to the vector sequence.
Thanks. Alejandro Martin and Ahmed Dhamad
But in case of Eukaryotic expression, does the stop codon is enough at the end of the gene or poly A signal is required?
Mahmoud El-awady , it all depends on the specific vector you have in mind. Most eukaryotic expression plasmids already carry the required regulatory hardware (promoter, binding sites for TFs, translational initiation (i.e. Kozak), polyadenylation, etc) so you only have to insert the coding sequence. What vector are you going to use?
Yes, the stop codon is enough to terminate the translation. The termination sequence in the vector will help to de-attache of the polymerase.
Thanks again Alejandro Martin and Ahmed Dhamad
Alejandro Martin There is no certain victor in mind, that's why i am taking caution.
Please all, i am still confused about this points:
1- Can the expression vector carry more than one cassette , each cassette with its own promoter?
2- Regarding placing the protein in-frame with vector, does this means that i cant place the gene of interest in another location via site-directed mutagenesis and corresponding restriction enzymes?
Thanks
Mahmoud El-awady,
1- Yes, definitely.
2- It depends. If you are going to clone the CDS in frame with a starting codon then no, you cannot go blindly changing things, but if the CDS carries its own starting and stop codon you have a bit more leeway.
Hope it helps!
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