TY - JOUR
T1 - Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant e protein ligand
AU - Reyes-Del Valle, Jorge
AU - Del Angel, Rosa M.
PY - 2004/3/1
Y1 - 2004/3/1
N2 - 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 45kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45kDa 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 45kDa, 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.
AB - 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 45kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45kDa 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 45kDa, 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.
KW - Affinity chromatography
KW - Putative dengue virus receptor
KW - Recombinant E protein ligand
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U2 - 10.1016/j.jviromet.2003.10.014
DO - 10.1016/j.jviromet.2003.10.014
M3 - Article
C2 - 14715312
AN - SCOPUS:0346727181
SN - 0166-0934
VL - 116
SP - 95
EP - 102
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -