Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand.
Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.
Reyes-del Valle
Jorge
J
Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Col. San Pedro Zacatenco, México City 07360, Mexico.
del Angel
Rosa M
RM
D000330:Aedes; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D003716:Dengue Virus / Q000254:growth & development / Q000502:physiology*; D004926:Escherichia coli; D005814:Genes, Viral; D008024:Ligands; D008970:Molecular Weight; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D011991:Receptors, Virus / Q000737:chemistry* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D012367:RNA, Viral; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C111476:dengue virus receptor; C072281:E protein TH Sman, Dengue virus