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Expression of β-agarase I DagA in prokaryotic cell and its activity identification

Published online by Cambridge University Press:  30 October 2009

Zhou Yan-Sheng ,
Wang Bao-Li  and
Qu Dong
Zhou Yan-Sheng
Affiliation:
College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100China
Wang Bao-Li*
Affiliation:
College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100China State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau, Northwest A & F University, Yangling, Shaanxi 712100China
Qu Dong
Affiliation:
College of Resources and Environment, Northwest A & F University, Yangling, Shaanxi 712100China
*
*Corresponding author. E-mail: wbl@nwsuaf.edu.cn
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Abstract

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.

Keywords

β-agarase I DagAprokaryotic expressioninclusion body renaturationbiological activity
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 2 , August 2009 , pp. 153 - 159
Copyright
Copyright © China Agricultural University 2009

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