title: Cloning, expression, and characterization of salivary apyrase from Aedes albopictus.
abstract: Apyrases (ATP diphosphohydrolase) hydrolyze the phosphodiester bonds of nucleoside tri- and diphosphates to orthophosphate and mononucleodides. They can inhibit platelet activation by depletion of adenosine diphosphate. In the current study, the Escherichia coli expression vector pET-19b equipped with an N-terminal histidine tag was applied to express the apyrase of Aedes albopictus. The gene-coding mature apyrase protein was amplified by RT-PCR and cloned into pET-19b. Soluble apyrase protein with high purity was successfully obtained by utilization of the suitable renaturation approach and Ni-NTA purification column. Four monoclonal antibodies to apyrase from A. albopictus were produced in male BALB/c mice immunized with the renatured apyrase. Using immunofluorescence assay and immunoblotting analysis, recombinant apyrase showed fine consistency with native apyrase. From kinetic analysis, it had a K (m) of 11.6 μM and V (max) of 1.02 nM/S/μg protein for adenosine triphosphate. Adenosine diphosphate-induced platelet aggregation was inhibited by approximately 6% when 0.4 μM recombinant apyrase was added and by about 9.5% when the concentration of recombinant apyrase was 0.8 μM. The effect on platelet aggregation was dose dependent. In conclusion, the apyrase of A. albopictus was cloned and expressed highly in the E. coli expression system. Recombinant apyrase protein showed biological activity, and anti-apyrase monoclonal antibody was also prepared.
authors.affiliation: Department of Microbiology and Parasitology, Shanghai Medical College of Fudan University, No. 138 Yixueyuan Road, Shanghai 200032, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Dong
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Fu
authors.forename: Xueping
authors.initials: X
authors.lastname: Li
authors.forename: Jianguo
authors.initials: J
authors.lastname: Jiang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Sun
authors.forename: Xunjia
authors.initials: X
authors.lastname: Cheng
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000330:Aedes / Q000201:enzymology* / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D001081:Apyrase / Q000235:genetics* / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D000911:Antibodies, Monoclonal; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; D000255:Adenosine Triphosphate; D001081:Apyrase