A time- and cost-efficient system for high-level protein production in mammalian cells

TLDR

Most proteins for structural biology are produced by high‑level expression in E. coli, but prokaryotic systems often fail to yield correctly folded functional proteins, so eukaryotic systems such as yeast and baculovirus‑infected insect cells are preferred. The study reports protocols for a simple, fast, and affordable method for transient protein expression in mammalian cells. The approach uses transient transfection of mammalian cells, providing a straightforward, rapid, and cost‑effective protocol.

Abstract

Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline.