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| Cloning and Expression of the Key Enzyme Gene in Biosynthesis of Sialyl Lewis X |
| YAO Jing1,2, REN Jing2, WU Zheng-jun2, SUN Ke-jie2, GUO Ben-heng1,2 |
1. College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China;
2. State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy Co., Ltd., Shanghai 200436, China |
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Abstract Sialyl lewis X (Slex), one of the common glyco ligands of the selectin family, restrains inflammation reaction by combining with the inflammatory cells competitively. Clone and expression of the key enzymes in the biosynthesis process of Slex could make its biosynthesis in vitro, and some related mastitis therapy research possible. Beta 1, 4 galactosyltransferase (GT) was the very one of the key enzymes in the biosynthesis process. In order to understand some related physicochemical property of GT gene, the gene sequence was analyzed by using approaches of bioinformatics online. The synthetic CDS of this gene was inserted into a recombinant plasmid pMD18-T, and subcloned to the expression plasmid pPIC9K later. The linear expression plasmid pPIC9K-GT was integrated to the genome of P. pastoris GS115 by electrotransformation. After inducible expression, the soluble target protein was detected by SDS-PAGE. It was proved that this gene could be expressed successfully in P. pastoris GS115. The phenol red method was used to determine the activity of the unpurified enzyme, and the specific activity was 16.4U/ml. This might be the foundation work for the further study.
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Received: 20 September 2011
Published: 25 December 2011
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