Optimizing K279a Protease Activity

We have previously shown that Dulbecco's modified essential medium (DMEM) low glucose (5.6 mM) medium (Invitrogen) is the optimal growth medium for inducing K279a protease activity (20 (link)). To prepare a stock solution of K279a CS, 10 μL of an overnight K279a culture was inoculated in 6 x 15 mls of DMEM low glucose (5.6 mM) medium and grown for 48 h at 37°C on an orbital shaker. K279a CS was passed sequentially through 0.45-μm and 0.2-μm filters millex filters (Millipore Corporation, Bedford, MA). Culture supernatant (90 mls) was then concentrated using 5-kDa nominal-weight limit (NMWL) cut-off Amicon® Ultra-15 filter devices (Millipore Corporation, Bedford, MA). All concentrates were centrifuged at 4,000 × g and subsequently diafiltered by centrifugation with sterile DPBS to remove any low molecular weight contaminants including glucose and amino acids present in DMEM. An equivalent volume of DMEM was used as a negative control and for correction during protein quantification using the BCA (bicinchoninic acid) assay.