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Escherichia coli BL21(DE3) is a commonly used protein production strain of the E. coli bacterium. This strain combines several features that allow for excessive expression of heterologous proteins . It is derived from the B lineage of E. coli .
The expression vector is transformed into one of several relevant strains of E. coli, most frequently BL21(DE3). The E. coli cell also has its own chromosome, which possesses a gene that is expressed to produce T7 RNA polymerase.
Typically, a sterile, filtered 1 mol/L solution of IPTG is added 1:1000 to an exponentially growing bacterial culture, to give a final concentration of 1 mmol/L. The concentration used depends on the strength of induction required, as well as the genotype of cells or plasmid used.
Cells are typically rod-shaped, and are about 2.0 μm long and 0.25–1.0 μm in diameter, with a cell volume of 0.6–0.7 μm 3. [19] [20] [21] E. coli stains gram-negative because its cell wall is composed of a thin peptidoglycan layer and an outer membrane.
Cell lines used for this system include: Sf9, Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni cells, and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells. [23] [25] With this system, cells do not lyse and several cultivation modes can be used. [23] Additionally, protein production runs are reproducible.
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Furthermore, lysozyme can serve as a tool in the expression of toxic recombinant proteins. Expressing recombinant proteins in BL21(DE3) strains is typically accomplished by the T7-RNA-polymerase. Via IPTG induction, the UV-5 repressor is inhibited, leading to the transcription of the T7-RNA-polymerase and thereby of the protein of interest.
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