a1 College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100 China
a2 State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau, Northwest A & F University, Yangling, Shaanxi 712100 China
a3 College of Resources and Environment, Northwest A & F University, Yangling, Shaanxi 712100 China
The DagA gene and DagA(), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA() were respectively expressed in Escherichia coli ER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA()-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.
(Received March 17 2008)
(Accepted May 27 2008)
First published Journal of Agricultural Biotechnology 2009, 17(1): 132–137