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Expression of human granulocyte‐macrophage colony‐stimulating factor gene in insect cells by a baculovirus vector
Author(s) -
Chiou Chuang-Jiun,
Wu Ming-chi
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80020-j
Subject(s) - sf9 , recombinant dna , microbiology and biotechnology , nuclear polyhedrosis virus , autographa californica , biology , western blot , spodoptera , recombinant virus , virology , tunicamycin , virus , transfection , baculoviridae , cell culture , gene , biochemistry , genetics , unfolded protein response
A plasmid pAc373GM‐CSF was constructed and co‐transfected into Spodoptera frugiperda (Sf9) cells with wild‐type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The recombinant virus vAc373GM‐CSF was identified and purified by several rounds of plaque hybridization. By assaying the culture medium, we demonstrated recombinant virus infected Sf9 cells expressing hGM‐CSF. Recombinant hGM‐CSFs with apparent molecular masses of 14.5, 15.5 and 16.5 kDa were detected by the Western blot method. All 3 forms have biological activity of hGM‐CSF. Following N ‐glycanase treatment, a single band of 14.5–15.5 kDa appeared in SDS‐PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 kDa species. Thus, the signal sequence of recombinant hGM‐CSF could be recognized and cleaved by infected insect cell and the resultant molecule secreted into the media.