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[ccp4bb]: semet-labelled protein question
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We are attempting to produce semet-labelled protein using the M9/
glucose approach (as done by Van Duyne/Clardy) and BL21 cells.
However, we find that our cells are not able to grow past an OD600 of
0.1 on this minimal media. Such a low density of cells translates
into a very low yield of protein for us and we are looking to increase
the density of cells before inducing. Initially, I thought that
glucose was the limiting factor and increased the glucose in the
minimal media from 0.5% to 5%. However, again, the cells would not
grow past an OD600 of 0.1. It seems that something else is limiting
cell growth. Might anyone have any suggestions on how to modify this
minimal media to get improved cell growth without comprimising the
ability to get semet incorporation? Any ideas are appreciated.
I should also mention that the protein is an NTPase and therefore
its expression has a certain metabolic cost.
Thanks in advance.