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| Cloning, Expression and Identification of Gene Encoding the β-Galactosidase of Streptococcus suis serotype 2 |
| ZHANG Feng-yu1,2, HU Dan1, GONG Xiu-fang1, ZHENG Feng1, PAN Xiu-zhen1, WANG Chang-jun1,2 |
1. Research Institute for Medicine of Nanjing Command, Nanjing 210002, China;
2. School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China |
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Abstract Objective:To clone and prokaryotically express the β-galactosidase of Streptococcus suis serotype 2 and to determine the enzymatic properties of the recombinant protein. Methods: bgaC gene was amplified by PCR using the primers which are on the basis of 05ZYH33 genome sequences and cloned into the expression vector. Thereafter, the gene was cloned into prokaryotic expression plasmid pET28a, and the recombinant plasmid pET28a-bgaC was transformed into E.coli BL21. After the induced expression by IPTG, the isolated BgaC protein was analyzed with SDS-PAGE and purified by chromatography. Thus obtaining the completely purified BgaC protein and its enzymatic activity was measured afterward. Results: bgaC gene could express highly in E.coli. The molecular weight of the recombinant expressed β-galactosidase BgaC was about 69kDa, the enzymatic activity analysis indicated the optimum temperature, action time, pH and the substrate concentration were 42℃,30min, 5.5 and 10mmol/L, respectively. Enzymatic activity of BgaC was about 1615U/ml, specific activity was 1076U/mg. Conclusions: The experimental results showed that the bgaC gene can be highly expressed in prokaryotic system, and the recombinant protein has the best enzymatic activity in optimizal temperature, reactive time and pH.
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Received: 08 November 2013
Published: 25 February 2014
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