Expression,Purification And Crystallization Of Sialidase From Porphyromonas Gingivalis TDC60

Periodontal disease is the most common oral disease in the world.As a part of human oral microbiota,Porphyromonas gingivalis(P.gingivalis)is a Gram-negative anaerobe,which plays a very important role in the occurrence and development of periodontal disease.Sialidase is one of the virulence factors of Porphyromonas gingivalis.Existing studies have shown that Porphyromonas gingivalis sialidase(PG.Sia)can modify glycoconjugates on the surface of host cells,thus exposing potential binding sites recognized by bacteria PG.Sia-catalyzed desialized protein can be further hydrolyzed by protease to generate peptides required for the degradation characteristics of Porphyromonas gingivalis.Given the importance of PG.Sia to the pathogen-host interaction and virulence,PG.Sia has sufficient potential to become a therapeutic target for periodontal disease.However,the lack of structural information hindered the further research and inhibitor design of PG.Sia.To obtain the structure of Porphyromonas gingivalis sialidase protein and explore its function,this experiment synthesized the sialidase gene of Porphyromonas gingivalis TDC60 strain with full gene,and successfully constructed the recombinant expression vector of Porphyromonas gingivalis sialidasep ET28 a using molecular cloning technology.After exogenous expression and screening of the recombinant expression vector in Escherichia coli system,purification and optimization of nickel column ion affinity chromatography and gel filtration chromatography,sufficient,uniform,and stable target proteins were obtained for protein crystallization experiments.After screening and optimizing the growth of protein crystals using protein crystallization kit,we found large,relatively complete,and well-shaped needle crystals under four high-salt conditions.The resolution of X-ray diffraction data collected under the conditions of 0.1 M Tris-HCl p H 7.0 and 1.4 M diammonium tartrate is 2.1 ?.Then the three-dimensional structure of protein and the construction of amino acids are analyzed,revealing the typical carbohydrate and catalytic domains.After that,the product sialic acid was simulated into the active site pocket,and combined with kinetic analysis,we could clearly identify the key residues required for substrate binding and catalysis.In addition,the structural comparison with other sialidases reveals the different characteristics of the active site pocket,which may give the specificity of substrate binding.These findings provide a structural basis for the further design and optimization of effective inhibitors targeting PG.Sia to fight oral diseases derived from Porphyromonas gingivalis.