Automation of large scale transient protein expression in mammalian cells

Yuguang Zhao,A Radu Aricescu,Susan Daenke,Jordan E Clay,Weixian Lu,E Yvonne Jones,Benjamin Bishop,Christian Siebold,Margaret Jones,David I Stuart

doi:10.1016/j.jsb.2011.04.017

Abstract

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.

Highlights

Fewer than 3% of the protein structures currently deposited in the Protein Data Bank (PDB) were solved using samples produced in mammalian cells (Nettleship et al, 2010), despite the recent emergence of rapid and cost-effective transient expression methods (Aricescu et al, 2006; Durocher et al, 2002; Meissner et al, 2001)
We present here simple and reproducible protocols for the automated large-scale transient transfection of constructs cloned into the pHLsec vector (Aricescu et al, 2006) in two human cell lines: human embryonic kidney (HEK) 293T and the N-glycosylation deficient HEK 293S GnTIÀ (Reeves et al, 2002)
A further advantage over the manual roller bottle system is that due to the low volume of media required in the roller bottles, HEK 293T cells in the presence of kifunensine typically survive for only 4 days post transfection while in HYPERFlasks efficient gas exchange, allowed by the permeable membrane on which the cells grow, results in cells producing proteins for up to 10 days posttransfection

Summary

Introduction

Fewer than 3% of the protein structures currently deposited in the Protein Data Bank (PDB) were solved using samples produced in mammalian cells (Nettleship et al, 2010), despite the recent emergence of rapid and cost-effective transient expression methods (Aricescu et al, 2006; Durocher et al, 2002; Meissner et al, 2001). The problems encountered for the large-scale expression of many proteins of eukaryotic, especially human, origin have increasingly focused attention on mammalian cell-based expression systems as a route for the production of targets that prove challenging in heterologous prokaryotic and insect cell expression hosts. In mammalian expression systems the protein, or complex, can be expressed in its native cell type, under physiological conditions, with numerous molecular systems working together for efficient production and quality control. The large-scale expression of eukaryotic proteins in a stable and soluble form is still a major bottleneck. This is due, in part, to the time consuming process required to maintain and expand cells, and to perform transient transfections.

Methods

Results

Discussion

Conclusion