The Single Step KRX Competent Cells: Efficient Cloning and High Protein Yields

We describe a new strain of Escherichia coli that has advantageous features for cloning and screening plus engineered attributes for tightly controlled protein expression. This new KRX strain is compatible with blue/white screening and can be made highly competent. In addition, KRX allows T7 RNA polymerase-based protein expression, one of the most widely used expression systems due to its well-defined promoter and the rapid elongation rate of the polymerase. In this strain, the T7 RNA polymerase gene is controlled by a rhamnose promoter. When we used the KRX strain to express firefly luciferase protein, the precise control of the rhamnose operon resulted in a dramatic induction ratio of 1,700-fold upon addition of rhamnose, whereas the leaky IPTG-inducible T7 RNA polymerase-based system in BL21(DE3)-derived strains only showed an 8- to 43-fold induction ratio, primarily due to high pre-induction levels of protein expression. Protein expression levels in KRX for three additional proteins were shown to be as high, or higher than, levels in BL21(DE3)-derived strains.

Promega Notes 94, 27–30.