Generation of Tetracycline-Inducible Mammalian Cell Lines by Flow Cytometry for Improved Overproduction of Membrane Proteins

Abstract

Overexpression of mammalian membrane proteins in mammalian cells is an effective strategy to produce sufficient protein for biophysical analyses and structural studies, because the cells generally express proteins in a correctly folded state. However, obtaining high levels of expression suitable for protein purification on a milligram scale can be challenging. As membrane protein overexpression often has a negative impact on cell viability, it is usual to make stable cell lines where the protein of interest is expressed from an inducible promoter. Here we describe a methodology for optimizing the inducible production of any membrane protein fused to GFP through the isolation of clonal cell lines. Flow cytometry is used to sort uninduced cells and the most fluorescent 5 % of the cell population are used to make clonal cell lines.

Structure–Function Relationships of Olfactory and Taste Receptors

“…Once it has been decided which strategy to use, the receptor needs to be expressed purified and crystallised, and each stage needs careful optimization. Overexpression systems using either insect cells or mammalian cells are the most successful, such as the Baculovirus expression system (Saarenpää et al 2015), the use of stable inducible cell lines (Andréll et al 2016) or the BacMam expression system (Goehring et al 2014). Green fluorescent protein fused at the C-terminus of GPCRs is a well-established method for rapid and efficient screening of expression levels, and for distinguishing folded versus unfolded receptor by a combination of fluorescence size exclusion chromatography (FSEC), differential solubilisation and confocal microscopy (Thomas and Tate 2014).…”

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

“…Combined, these control samples serve to properly set photomultiplier tube (PMT) detector voltages, and to calculate the fluorescence compensation matrix.Fluorescence-minus-one (FMO) control samples are cells expressing all but one of the fluorochromes. They ensure that spread of fluorochromes into the channel of interest is properly identified and are ideal for determining accurate gating boundaries.Viability dyes such as propidium iodide (PI), DRAQ7, 7-AAD or TO-PRO-3 that penetrate dead and dying cells and bind their DNA can be used to design a gating strategy to remove these cells from the transduced cell population, or to monitor cell viability to decide on the appropriate time for harvesting of expression cells or conditioned medium.When using inducible cells, FACS can be performed using either the uninduced 65 or the Dox-induced fluorescence. Importantly, the 405 nm laser readily excites Dox, introducing potential bias in fluorescence measurements.…”

“…When using inducible cells, FACS can be performed using either the uninduced 65 or the Dox-induced fluorescence. Importantly, the 405 nm laser readily excites Dox, introducing potential bias in fluorescence measurements.…”

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

“…In this work we report the expression of the wild type CB 2 in mammalian cell cultures. An increasing number of successful examples of GPCR expression in mammalian cells have been published recently 14,24,25 . These include serotonin receptor 5HT3A expressed in tetracycline-induced HEK293S-TetR cells (about 1.7 mg/L of culture) 26 and olfactory receptor 17-4 expressed in HEK293S-GNTI-cultivated in bioreactor (3 mg/L of culture) 27 .…”