As shown in Figure 2, Lemo21(DE3) makes it possible to identify gene expression levels for the optimal production of any protein using one strain and a limited number of culture/induction conditions. Importantly, at the optimal rhamnose concentration the population of cells in the culture remains completely homogenous; i.e., all cells produce the target protein.
In Figure 2A the production of the polytopic membrane protein YidC was optimized in Lemo21(DE3). To monitor production levels of functional membrane protein material, green fluorescent protein (GFP) was fused to the C-terminus of YidC. Protein GFP-fusions allow the use of whole cell fluorescence to rapidly and accurately monitor protein production levels.
Cells were cultured in Lysogeny Broth (LB) in the presence of different concentrations of rhamnose, and the expression of the gene encoding the T7 RNA polymerase was induced with 0.4 mM IPTG. In Figure 2B the production of a set of proteins in BL21(DE3) and Lemo21(DE3) was compared.
The target proteins are the bacterial polytopic membrane proteins EnvZ, PheP, and YidC; the human polytopic membrane proteins Tetraspanin (Tsp) A and B; the soluble proteins Glutathione S-transferase (GST) and GFP alone. All proteins, except GFP itself, were C-terminally fused to GFP.
Cells were cultured in LB medium and whole cell fluorescence was measured 8 hours after induction with 0.4 mM IPTG. For protein production in Lemo21(DE3), the optimal rhamnose concentration was used. Notably, fluorescence values of TspA and TspB were multiplied by 10, and fluorescence values of GST-GFP and GFP were divided by 10 and 50, respectively.
In Figure 2C, an example of the optimization of the production of a disulfide bonded single-chain variable fragment (scFv) in the periplasm of Lemo21(DE3) is shown. To guide the scFv to the periplasm it was equipped with a signal sequence (p = precursor protein, localized in the cytoplasm), which is clipped off upon translocation of the protein into the periplasm (m = mature protein, localized in the periplasm).
Notably, the scFv produced at the optimal rhamnose concentration was functional.
Taken together, the versatility of Lemo21(DE3) makes it possible to rapidly identify the optimal conditions for the production of any protein using one strain and a limited number of culture/induction conditions.