Cloning,Heterologous Expression And Characterization Of A Novel N-glycosidase PNGase H~+

Glycomics is becoming a new hot spot of biology research after genomics and proteomics.Glycoprotein has been found to play important roles in diverse biological processes and has gained the most extensive studies.Endo-N-glycosidase(PNGase)is the tool enzyme which enables the release of N-glycans from the glycoprotein for further study.It is widely used in the research on sugar chain structure information,relationship between its structure and function,effects of sugar chain on glycoproteins' function,and protein structure analysis and so on.The currently available commercial N-glycosidase mainly includes two different types:PNGase F and PNGase A.Both of the enzymes have unavoidable disadvantages when used for protein deglycosylation,and therefore cannot fully meet the needs of the rapid development of glycomics.It is thus necessary to discover a novel endo-N-glycosidase which has the advantages of both PNGase F and PNGase A,and at the same time lacks their disadvantages.In details:the novel endo-N-glycosidase should not have its own glycosylation,can be expressed in prokaryotic system and can be used to deglycosylate different types of glycoprotein to the greatest extent.Based on our prelimitary gene mining work,a novel PNGase enzyme encoded gene was discovered from the bacterial strain Terriglobus roseus DSM 18391.In this study,the gene cloning,recombinant expression of the cloned gene and enzyme characterization are presented.1.A new type of endo-N-glycosidase PNGase H~+ gene was discovered by gene mining and cloning from acidobacterium Terriglobus roeus DSM 18391.By ligating the PNGase H~+ gene to a pET30a vector and transforming into E.coli BL21(DE3),the PNGase H~+ engineering bacteria were restructured.Through conducting inducible expression of PNGase H~+ in specific foster conditions,and making purification through the Ni-NTA affinity chromatography,the PNGaes H~+ enzyme with high purity was obtained.2.In order to detect the activity of the recombinant protein,fluorescently-labeled GNGN was used as the standard substrate,and a UHPLC-based method was established for activity detection.It was found that the recombinant protein has the activity similar to PNGase A,with the optimum pH of 2.6 and optimum reaction temperature of 30 ?.No metal ion was required by PNGase H~+ for its full enzymatic activity.Most of the denaturants detected have the inhibitory effect on the enzyme activity.3.It was found that PNGase H~+ can function like PNGase F to the high Mannose type,composite type and hybrid-type of N-linked glycans derived from mammals.It can also function like PNGase A to plant-derived N-linked glycans which contains core?1,3-fucose.Moreover,differing from PNGase A which can only deglycosylate glycopeptide,the novel PNGase H~+ can work directly on glycoproteins.It was also more efficient than PNGase F when worked on the HRP N-glyans without core ?1,3-fucose.In genral,a novel PNGase H~+ originated from Terriglobus roseus was successful discovered and recombinantly expressioned.It has all the advantages of both PNGase F and PNGase A,while at the same time is lack of any disadvantage of the two.The discovery of such a novel endo-N-glycosidase provides a more powerful,economical and efficient tool for glycobiology research.