title: Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene.
abstract: To increase the selection efficiency and productivity of stable clones expressing recombinant antibody-IL-2 fusion protein, a dicistronic expression vector containing the anti-erbB2 scFv-Fc-IL-2 fusion gene followed by a weakened neo gene, was constructed to allow for the concurrent translation of the recombinant protein and mutated neomycin phosphotransferase from a single mRNA. The presence of the mutant enzyme in the transfectomas resulted in a decreased resistance of cells in the presence of an elevated level of G418 and retarded cell growth. The transfectomas containing the mutant enzyme expressed considerably higher levels of the fusion protein than those containing the normal enzyme. Furthermore, these positive clones had an almost identical level of recombinant gene expression, which was very stable even when the concentration of G418 was significantly increased. Thus, the selection efficiency of strongly positive producers was remarkably increased. Our results demonstrate that the dicistronic expression vector is useful for the selection of highly expressing clones. Combined with an amplification system, this vector may have potential usage for the expression of recombinant antibody and the yield may be further improved by this expression system.
authors.affiliation: Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, PR China.
authors.forename: Liyong
authors.initials: L
authors.lastname: Chen
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Xie
authors.forename: Yan
authors.initials: Y
authors.lastname: Teng
authors.forename: Minggao
authors.initials: M
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Shi
authors.forename: Lu
authors.initials: L
authors.lastname: Qian
authors.forename: Meiru
authors.initials: M
authors.lastname: Hu
authors.forename: Jiannan
authors.initials: J
authors.lastname: Feng
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.forename: Beifen
authors.initials: B
authors.lastname: Shen
authors.forename: Ning
authors.initials: N
authors.lastname: Guo
mesh_terms: D000818:Animals; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D019868:Kanamycin Kinase / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D019868:Kanamycin Kinase