title: Large-scale production of functional recombinant CAR in a baculovirus- insect cell system.
abstract: Coxsackievirus group B and adenovirus receptor (CAR) is a major receptor for the adenovirus groups that has drawn overall attention over the past decade. Although this protein could potentially be used as an agent for the blocking of adenovirus infection, large-scale production of highly purified human CAR in eukaryotic expression system has not been reported. In the present study, we showed the construction of recombinant baculovirus highly-expressing the extracellular domain of human coxsackievirus-adenovirus receptor (exCAR) in High Five insect cells. The recombinant exCAR was recovered from the cell culture medium as a secreted soluble protein and purified by Ni-NTA affinity chromatography. The final yield of recombinant exCAR was about 8-10 mg/l of supernatant with the purity of 96.3%. Binding activity assay showed that the recombinant exCAR exhibited an intact ability of binding to the knob domain of the adenovirus type 5 fiber protein (Ad fiber knob) displayed by T7 phage. These results showed that the recombinant human exCAR produced in insect cells and purified by Ni-NTA chromatography retained its ability to bind to the Ad fiber knob and could potentially be used in therapy of adenovirus infection.
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji Jospital, Sahnghai Jiaodong University School of Medicine, Shanghai Cancer Institute, China.
authors.forename: M
authors.initials: M
authors.lastname: Zhou
authors.forename: B
authors.initials: B
authors.lastname: Shi
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Gu
authors.forename: Z
authors.initials: Z
authors.lastname: Li
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
chemical_list: D011991:Receptors, Virus; D011994:Recombinant Proteins; C085301:adenovirus receptor