I am working on a project where I have cloned and overexpressed an
aminotransferase in E. coli. We are trying to use crude extracts of the
recombinant strain in a bioconversion with a dipeptide as substrate. It
appears that the reaction is taking place but the product is then being
cleaved into its constituent amino acids. The longer after induction the
culture is taken, the worse this problem is. (The system uses a minimal
medium and L-tryptophan as inducer.) My guess is that the burden of
overproducing the aminotransferase is causing a stress response (which
might include a protease). I have both ampicillin- and
kanamycin-resistant versions of the expressing plasmid and the degradation
is decidedly more pronounced in the latter.
My questions:
1. Are there any known E. coli strains that lack a protease which would
attack a dipeptide?
2. We are currently running our fermentations at 30oC. Would going to a
lower temperature help alleviate our problem? How about using a richer
medium?
3. Any other hosts (bacterial or yeast) which have low protease activities?
Any input would be welcome. Thanks.
Steve
