Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins

Abstract

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production to milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this Protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting, and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability, lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks, and routinely achieve a ~3-10-fold improvement in protein production yield per cell as compared to transient transduction or transfection.

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

“…In an academic context, mammalian production of PTKs seems mostly limited to small-scale expression from transiently transfected cells (Deng et al, 2014). Nevertheless, recent lentiviral transfection protocols (Elegheert et al, 2018) or baculovirus-mediated gene transfer into mammalian cells (BacMam) (Goehring et al, 2014;Hofmann et al, 1995) obviate the generation of stable cell lines and shorten the mammalian expression protocol to a time frame that is comparable with insect cell expression. This method is thus also becoming attractive for the large-scale expression of PTKs.…”

Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses

“…A major disadvantage is the time required to develop a monoclonal cell line (Chaudhary, Pak, Gruswitz, Sharma, & Stroud, 2012). Stable cell lines created through lentiviral transduction have been shown to ameliorate this disadvantage (Elegheert et al., 2018). Baculovirus transduction of mammalian cells (BacMam) is a powerful technique to express proteins.…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…An alternative approach increasingly applied as a gene delivery methodology for protein production is based on the use of lentivirus, owing to their ability to transduce a broad range of cell types (Bandaranayake and Almo 2014). Aiming to combine the ease and speed of transient transfection with the robust expression of stable cell lines, Elegheert et al (2018) constructed a lentiviral plasmid suite around the transfer plasmid pHR-CMV-TetO 2 that is designed for large-scale protein expression from HEK293 cell lines and allows subcloning of cDNA from the plasmid PHLsec usually applied for transient transfection. This approach was tested in both soluble and MP, and in general, the typical lead time for protein production using this strategy is of 3-4 weeks and approximately three to tenfold improvement in protein production yield per cell was obtained, in comparison with transient transfection (Elegheert et al 2018).…”