I have read some articles, in which they have used BL21 and in other some cases it is M15 cells. So, which is the better host strain for expression and why ?
Dear Santhosh
I have limited practical experinces with T5 promoter becuase in my hands it showed low performance respect T7 or pBad. In theory differently to the T7 promoter (which is not recognized from the E.coli RNa polimerase and require specific strains codifing for the T7 RNA polimerase, as (DE3) strains or T7 express strains) the T5 promoter is recognized for E.coli polimerase so in principle you can use any E.coli strains. This means that is certanily possible use BL21(DE3) also if the presence of the DE3 is needless. Is it possible that this will not be the perfect strain, becuase its has a metabolic bost due to the production of T7 polimerase, but if you have this strain you can certanly test it. The M15 strain is a strain containg an addictional plasmid that allow better repression before induction and therefore low basal expression but probably there is not a universal best strain, it dependes from protein to protein. For example in case of disulfide bridge, Origami could be usefull.
good luck
manuele
I dont have information about this pQE30 vector, but if it carries a strong bacterial promoter, T7 promoter, DE3 strain can give you good expression results.
If you are using pQE-30 vector with BL21 cells, you´ll need to transform them with pREP4 as well. pREP4 carries lac I gene to produce lac repressor. This will prevent your system from leaking expression. (M15 cells already have pREP4).
Check http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAexpressionist_EN.pdf
for more information. Cheers
m15 is the better host for pQE30 as it contains T5 promotor and M15 also supports the same. but in case of BL21 as it supports T7 promotor, I can say this wont be the better host than M15.
We are going to make a construct with T5 promoter. We are going to order M15 strain from Qiagen. However, I could not find the product except one PDF file about it. Do they stop selling M15 strains? Do anyone know where are the other resources to get the strain? Thank you!
Update: I used several cell strains with no pREP4, transformed with pQE-60 vector. They all produced my protein fine. It is true that all E. coli polymerase can naturally recognize T5 promotor. I think it is also safe to guess that my protein is not "toxic" to the cells.
E coli strains carry lacIqΔM15 mutation on the F′ episome, which could express higher level of lacI repressor, would be options to evaluation.
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