Abstract
A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.
Highlights
Simultaneous cell surface display and secretion is desirable for protein evolution and selection
A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cellbased functional assays, to be used during the selection process
As a percent of all bands visualized, band SD3 (Fig. 2b) represents Ͻ10% of Ig1-1, 30% of Ig1-2, and ϳ50% of Ig1-3, indicating some modulation of alternative splicing could be accomplished. These data suggested that alternative RNA splicing was responsible for the simultaneous secretion and cell surface presentation of the antibody, a theory that was explored in further experiments described below
Summary
Simultaneous cell surface display and secretion is desirable for protein evolution and selection. A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cellbased functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Mammalian cell expression is the dominant technology for therapeutic antibody production, in vitro technology for isolation and engineering of
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