"BL21 (DE3)-RIL cells, which are compatible with the T7 promoter system, contain plasmids that encode arginine, isoleucine, and leucine tRNAs that are rare in E. coli, and are deficient in both lon and ompT proteases, which minimizes in vivo degradation of the target protein"
I just don't understand why the Prescence of these tRNAs in BL21 and their absence in lon and ompT proteases would minimize in vivo degradation of the target protein, can you further explain this?
A classic run-on sentence. There are two concepts mentioned. They should have been separated onto two sentences to avoid confusion.
1. The expression of tRNAs for rare codons, which improves expression of proteins from genes that contain rare codons.
2. The absence of two proteases, which helps to reduce the proteolytic degradation of the target protein.
The BL21 (DE3)-RIL cells described contain two separate alterations: (1) the contain plasmids that encode arginine, isoleucine, and leucine tRNAs that are rare in E. coli; and (2) are deficient in both lon and ompT proteases to minimizes in vivo degradation of the target protein.
Thanks, that was very helping.
I have a couple more questions.
1) I need an explanation of the following statement from a protein expression review article
" The only drawback of these vectors is that they typically have only a single restriction site for N-terminal cloning (NcoI). Thus, if you have a NcoI site in the DNA sequence of your “to-be-expressed” protein, then you cannot use these vectors for cloning unless they are genetically modified to incorporate a new restriction site. Because proteins often have multiple NcoI sites, including proteins we are studying in our laboratories, we modified a subset of these vectors to include an NheI site immediately following the NcoI site, allowing these modified vectors to be used to subclone DNA sequences with NcoI sites"
My question is why these vectors, which have just one NcoI cloning site, cannot be used to subclone DNA sequences with NcoI sites? and what the difference that the other site (Nhel) would make to fix this issue?
2) when to use a restriction enzyme that creates a blunt or sticky ends? I know the difference between these ends, but I do not understand why to use a restriction enzyme that creates one of these two specific ends?
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