Development of an Integrated Expression Platform for Protein Production in Eukaryotic Hosts

Abstract

A prerequisite for structural and biochemical analyses of proteins is the availability of large amounts of high-quality material. However, the recombinant production of complex proteins typically requires a profound screening process to identify expressible constructs. Furthermore, the evaluation of an optimal expression host has an important impact on protein quality, yield and cost efficiency. As a result, the production of target proteins in eukaryotic systems is a major bottleneck in structural biology. In this work, the novel expression/donor vector pFlpBtM has been constructed to be utilised for both, the rapid screening for expressible protein variants and the evaluation of an optimal expression host. The range of applications comprises a variety of highly efficient eukaryotic hosts used at the Helmholtz Protein Sample Production Facility (PSPF). It was realised by the unique combination of different expression methods including transient, viral and stable expression strategies. The efficiency of the vector is demonstrated in a comparative expression study of model proteins from different protein classes which have been produced by transient expression in HEK293-6E cells as well as in the baculovirus expression vector system and by stable expression using a recombinase mediated cassette exchange system in CHO Lec3.2.8.1 cells. Additionally, engineered BEVS host cell lines were generated, which enable the stable genomic expression of auxiliary proteins to facilitate the production of particularly challenging target proteins. The flexible RMCE system was implemented in insect cell lines for the first time to provide optimised coexpression for each target protein and deploy compatibility to the RMCE CHO Lec3.2.8.1 cell lines. With the combination of a versatile donor/expression plasmid and optimised mammalian and lepidopteran producer cell lines, the PSPF offers an integrated platform for protein production in eukaryotic systems.