I have a bacterial gene sequence code for a protein but it's start position/ codon is TTG. Is it a mutation or consider as a alternative start codon?. I need to express this protein in a BL 21 E.Coli host. Is it possible?
Beware that just because UUG can be used as a start codon in E. coli does not mean it is used as such in the bacterium from which the gene is derived. Alternative start codon usage can vary a bit accross the taxonomical spectrum (take a look at NCBI's genetic codes page). Also, for it to work as an alternative start codon it needs a properly spaced RBS; have you tried looking for one?
As Syied advices, if you are going to express this in E. coli it is better to replace the native start codon with AUG, otherwise the translation initiation rate (hence expression) is much lower.
Here is a helpful Wikipedia article about start codons.
https://en.wikipedia.org/wiki/Start_codon
This part is particularly relevant:
E. coli uses 83% AUG (3542/4284), 14% (612) GUG, 3% (103) UUG and one or two others (e.g., an AUU and possibly a CUG).
Well-known coding regions that do not have AUG initiation codons are those of lacI (GUG) and lacA (UUG) in the E. coli lac operon.
For over expression in BL21, I ll suggest you to replace the native start codon with AUG.
Beware that just because UUG can be used as a start codon in E. coli does not mean it is used as such in the bacterium from which the gene is derived. Alternative start codon usage can vary a bit accross the taxonomical spectrum (take a look at NCBI's genetic codes page). Also, for it to work as an alternative start codon it needs a properly spaced RBS; have you tried looking for one?
As Syied advices, if you are going to express this in E. coli it is better to replace the native start codon with AUG, otherwise the translation initiation rate (hence expression) is much lower.
These percentages are based upon data prior to 2000. I would recommend that you look at the data from Article An analysis of initiation codon utilization in the Domain Ba...
Unfortunately, the software is no longer on a active server, but is available for download at https://github.com/ajvilleg/Inidon
having the same issue here, did you replace the start codon with ATG? and did it work? Nilmini Kumari
thanks in advance
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