Cell-Based Protein Expression

Promega offers two strains of competent E. coli cells modified to improve transformation efficiency and to provide regulated or inducible expression of recombinant proteins: BL21(DE3)pLysS and Single-Step KRX competent cells.

BL21(DE3)pLysS is a derivative of BL21 that has the T7 RNA polymerase gene under the control of the lacUV5 promoter (inducible by IPTG). Single-Step (KRX) Competent Cells contain a chromosomal copy of T7 RNA polymerase driven by a rhamnose promoter (rhaBAD, inducible by rhamnose). Background protein expression levels in Single-Step (KRX) Competent Cells are significantly lower than those of BL21(DE3)-derived strains and therefore are recommended for expression of proteins that are toxic to
E. coli.

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What is Cell-Based Protein Expression?

Cell-based protein expression allows for high levels of recombinant protein production, using prokaryotic or eukaryotic cells. E. coli is a common host that is well-known for straightforward genetic manipulation and production of many different types of proteins. The most popular method for E. coli cell-based recombinant protein expressionuses a T7 expression host and an expression vector containing a T7 promoter. The gene of interest is cloned downstream of the T7 promoter and the expression vector is introduced into the host. An inducer, such as rhamnose or IPTG, is added to the culture to induce the T7 promoter. The gene of interest is then transcribed and translated by the host cell’s endogenous protein translation machinery.

Different promoters offer different strengths of control over gene expression. The rhamnose (rhaBAD) promoter offers tight control of the proteins expressed via a T7 promoter. This is ideal when recombinant protein products could be detrimental to the cell.