Using native hydantoinase promoter to induce D-carbamoylase soluble expression in Escherichia coli

By use of PCR, the genes encoding D-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coff BL21 (DE3) to overexpreSS D-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (PH,,) whose optimal length was confirmed to 209bp. Furthermore, the REIS region in the downstream Of PH111 was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E coli strain Topl0l". The enzyme activity of ToplOF'/pGEMT-R-DCB grown at 37 IC was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 oC. (c) 2008 Elsevier B.V. All rights reserved.