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From Robert Frank Standaert's Thesis: Consultation of
a biochemistry textbook indicated that threonine, isoleucine, and lysine inhibit
methionine biosynthesis in E. coli by inhibiting aspartokinases, and that
phenylalanine and leucine are synergistic with lysine. Incorporation
of this information into a strategy for SeMet labeling was simple. E. coli XA90 were grown in 1 L of M9
medium to mid-log phase (A600=0.6), and 60 mg of SeMet (sigma) along with
100 mg each of threonine, lysine hydrochloride, and phenylalanine and 50 mg
each of leucine, isoleucine, and valine were added as solids to the growing
culture. After another 15 min, IPTG was added to 1 mM, and the procedure
was completed as usual. Purification was performed
essentially as for native protein, except that DTT at a concentration of 5-10
mM was maintained throughout on the expectation that SeMet would be sensitive
to oxidation. The yield of protein was 10 mg, about what would be obtained
from a normal prep. I used to buy my SeMet from Acros in 1998.
It was 10 times cheaper than Sigma and it works. Reference: |
