Stabilized, long-term expression of heterodimeric proteins from tricistronic mRNA

A major problem in the use of recombinant mammalian cells for protein overe xpression is their long-term stability, in particular, when the foreign gen e product exerts a negative effect on the producer cells. We have addressed this issue and developed a Vector system for the stable expression of hete rodimeric recombinant proteins in mammalian cells. In this system, the two recombinant cDNAs and the puromycin-resistant gene are transcribed as a sin gle tricistronic transcript. An efficient translation of the internal cistr ons is mediated by internal ribosome entry sites between them. On the examp le of expression of a heterodimeric antibody fusion protein in BHK-21 cells , we show that the translational coupling of the antibody genes to the sele ctable marker in a tricistronic expression construct allows long-term stabi lization of expression by continuous application of selection pressure. Thi s vector system allows fast and straightforward construction of expression plasmids for the generation of producer cell lines, even for complex hetero dimeric proteins with unlimited long-term stability. (C) 2000 Elsevier Scie nce B.V. All rights reserved.