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| Expression of β-agarase ⅠDagA in Prokaryotic Cell and Its Activity Identification |
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Abstract dagA gene and dagA(▽) which is a dagA gene encoding sequence without signal peptide were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by PCR. After ligation with pET21 vactor, dagA and dagA(▽) were expressed in E. coli ER2566, respectively, using molecular chaperone DsbC and FkpA. Strain of ER2566- pET21a-dagA(▽)-DsbC was screened as high effective expressing system, in form of the inclusion body in which had the target protein up to 60 % of total bacterial protein. DagA protein was renaturated and purified by dissolving in 8 mol/L of urea, Ni-NTA resin affinity chromatography and refolding by urea gradient method. DagA was about 30.8 kD identified by SDS-PAGE and had the ability to digest agarose. At the range of pH 4.8 to 6.8, DagA could hold an bio-activity of beyond 60 %, with 5.8 as optimum pH value and its activity was available under 37 to 60 ℃, with 55 ℃ as optimum temperature.
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Received: 17 March 2008
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