Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene.
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.
Chen
Liyong
L
Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, PR China.
Xie
Zhigang
Z
Teng
Yan
Y
Wang
Minggao
M
Shi
Ming
M
Qian
Lu
L
Hu
Meiru
M
Feng
Jiannan
J
Yang
Xiao
X
Shen
Beifen
B
Guo
Ning
N
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
D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D019868:Kanamycin Kinase