反应详情

The E. coli (XLI Blue strain) transformed with recombinant pHLASP or wild type pBluescript SK− phagemid were grown overnight in 15 ml of Luria broth containing glucose (0.1%) and ampicillin (50 μg/ml) in the absence or presence of 20 mM IPTG. The bacteria were harvested, treated with 500 μL of 0.05 mg/ml lysozyme in 25 mM Tris.Cl pH 8.0, 10 mM EDTA and 50 mM glucose for 10 min at ambient temperature. The treated bacteria were diluted with ice cold 1.5 ml of 50 mM Tris.Cl, pH 8.0, 0.01% β-mercaptoethanol and 0.01% sodium azide and sonicated by three 10 sec bursts in ice cold conditions. The protein concentration in the bacterial sonicates was determined48. Aspartoacylase assays were carried out in duplicate with 450 μg protein of bacterial sonicate under standard incubation conditions described elsewhere16. Controls lacking substrate or enzyme during the incubation were run simultaneously to account for background absorption in each assay. One mU of enzyme activity is defined as 1 nanomole of aspartate released/min of incubation time.

1. concentrationThe protein concentration in the
2. custombacterial sonicates
3. customof bacterial sonicate under standard incubation conditions
4. customto account for background absorption in each assay