Earticle

초록

영어

Pasteurella m ultocida is a m ultifunctional sialyltransferase (PST) which transfer sialic acid from cytidine 5’-monophosphonuraminic acid (CMP-NeuAc) to an acceptor sugar. Multifunction of PST was controlled by temperature and pH. Multifunction of PST is associated with its conformational change and binding with lactose acceptor according to pH and temperature. By analyzing the protein-substrate binding structure, Arg313, Asn85 and Ser62 are selected for potential sites affecting lactose binding. We generated mutants library for each sites through the site directed saturation mutagenesis. And mutagenic primers were constructed by rational design. To identify combinatorial effect of 3 sites, multi-site mutagenesis method was used using the anchor sequence and gateway method. To select mutants having an increased activity, pH-color assay method is used. Screening is based on the color changes of the pH indicator cresol red when proton is released during the transfer of NeuAc from CMP-NeuAc to acceptor substrate. By using the method, mutants were selected and combinatorial mutagenesis was done using the selected amino acid sites.

키워드

저자

• Yun-Hee CHOI [ Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Republic of Korea. ]
• Joo-Hyun SEO [ Dept. of Chemical and Biological Engineering, Seoul National University. ]
• Min-Ho CHA [ Dept. of Chemical and Biological Engineering, Seoul National University. ]
• Dae-Hee KIM [ GeneChem Inc., Daejeon 302-804, Republic of Korea. ]
• Byung-Gee KIM [ Dept. of Chemical and Biological Engineering, Seoul National University. ]

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간행물

표지보기 관심저널 등록

• 간행물명

  한국생물공학회 학술대회

• 간기

  반년간

• 수록기간

  1985~2013

• 십진분류

  KDC 476 DDC 576