earticle

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.