short term and long term expression of target proteins and its kinetic properties with or without selected dlm
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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.
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