Molecular Clone and Expression of Phospholipase A1 Gene from Citrobacter youngae in Escherichia coli

Abstract:

For the heterologous expression of phospholipase A1，the gene of phospholipase A1 from Citrobacter youngae（CICC No.21596） was cloned into the expression vector pET28a（+） to construct the recombinant plasmid pET28a（+）-pla1.Then the recombinant plasmid was transformed into E.coli BL21（DE3） to achieve the recombinant strain pET28a（+）-pla1/DE3. After subsequent induction by isopropyl β-D-1-thiogalactopyranoside （IPTG），an approximately 33 000 protein was detected in the cell lysate supernatant by SDS-PAGE. The PLA1 activity was detected in an egg yolk agar plate，indicating that the PLA1 gene was expressed in E.coli. The best induction conditions for PLA1 expression were achieved by the optimization of fermentation condition and it was showed as follows，4% of inoculum amount，2 h of initial culture，8 h of induced culture in the presence of 0.4 mmol/L of IPTG，and growth at 37 ℃. Under the optimized conditions，the maximum PLA1 activity in the supernatant was （5.6±0.2） U/mL.