Application Note | January 7, 2014

Recombinant GPCR Production In A Stirred-Tank Bioreactor

Source: Eppendorf, Inc
AA314_HEK293_Cells_Protocol

By K. Christopher Min, Yan Jin, and Vikram Gossain

We have been studying rhodopsin with an interest in determining the conformational change that leads to signal transduction in this class of receptors. Although there has been some success in expressing select members of the large GPCR family in bacterial systems, the best characterized expression systems have generally been in mammalian tissue culture. In our case, we isolated stable cell lines in which the desired receptor is expressed upon exposure to tetracycline. The cell line was derived from HEK293 cells, which can be grown in suspension. Attempts to scale up production of recombinantly-expressed protein by the use of spinner flasks were unsuccessful.

Based on our initial experiments using tissue culture plates, we had expected approximately 1 mg of recombinant protein for 1 L of cells grown in suspension, but found that expression levels in spinner flasks were closer to 0.1 mg per L. Use of a stirred-tank bioreactor allowed for optimization of cell growth, as described below, and resulted in higher cell densities with concomitant higher levels of expression of our recombinant protein.

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