Stabilized, long-term expression of heterodimeric proteins from tricistronic mRNA.
A major problem in the use of recombinant mammalian cells for protein overexpression is their long-term stability, in particular, when the foreign gene product exerts a negative effect on the producer cells. We have addressed this issue and developed a vector system for the stable expression of heterodimeric recombinant proteins in mammalian cells. In this system, the two recombinant cDNAs and the puromycin-resistant gene are transcribed as a single tricistronic transcript. An efficient translation of the internal cistrons is mediated by internal ribosome entry sites between them. On the example of expression of a heterodimeric antibody fusion protein in BHK-21 cells, we show that the translational coupling of the antibody genes to the selectable marker in a tricistronic expression construct allows long-term stabilization of expression by continuous application of selection pressure. This vector system allows fast and straightforward construction of expression plasmids for the generation of producer cell lines, even for complex heterodimeric proteins with unlimited long-term stability.
Mielke
C
C
GBF - Gesellschaft für Biotechnologische Forschung mbH, Department of Gene Regulation and Differentiation, Mascheroder Weg 1, 38124, Braunschweig, Germany.
Tümmler
M
M
Schübeler
D
D
von Hoegen
I
I
Hauser
H
H
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D015870:Gene Expression; D005786:Gene Expression Regulation; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D013997:Time Factors; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D007074:Immunoglobulin G; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA