| Peptide- N4-(N-acetyl-p-glucosaminyl) asparagine amidase F (PNGase F, EC 3.5.1.52) removes glycan moieties from glycoproteins under relatively mild conditions. PNGase F has the broadest substrate specificity and can hydrolyzes all types of N-linked carbohydrate chains. It has been used as a powerful tool in analyzing the structual and biological functions of N-linked glycan glycoproteins. Currently, commercial PNGase F is mainly extracted from Flavobacterium meningosepticum. Purification of native PNGase F is a time-consuming, multistep process, which limits its utilization and large-scale preparation greatly.The gene of PNGase F from F. meningosepticum was cloned into plasmid pYDl, which allowed regulated expression, secretion and detection. The fact that the expression of PNGase F gene with its secretion signal peptide at the C-terminal of a-agglutinin in Saccharomyces cerevisiae led to the display of the protein on the extracellular surface of S. cerevisiae was confirmed by immunofiuorescence microscopy. Fluorescence Activated Cell Sorter (FACS) analysis indicated that, after 36 hours cultivation, 47.6% of the cell surface was covered with the fusion proteins. Activity measurement indicated that the enzyme was active and the enzyme activity reached the highest level after been induced 36h by 2% galactose. The optimum activity of the immobilized enzyme was obtained in the acetate buffer (pH5.5) at 37 ℃. This indicated that the immobilized enzyme on yeast cell surface could be used as a whole cell biocatalyst for producing complex carbohydrates from natural glycoproteins.In order to obtain soluble PNGase F, The gene of PNGase F was constructed to the vector pET28a. After PNGase F was expressed in the host cell, SDS-PAGE analysis revealed that the molecular weigh of expressed product of PNGase F was about 34kD. The expression level reached about 30% in E. coli. This recombinant PNGase F protein was purified by metal affinity resin and the purity is above 90%. |
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