High-Throughput Expression Screening in Mammalian Suspension Cells.

Abstract

Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. However, this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many parameters including truncations, point mutations, investigation of orthologs, fusion to peptide or protein tags at the N- or C-terminus, the coexpression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multiparallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multiparallel method to express proteins in mammalian suspension cells by transient transfection in 24-well or 96-well blocks.