KRX bacteria are a strain of E. coli engineered for cloning, screening and expressing proteins. The Single Step (KRX) Competent Cells are highly competent, allowing efficient transformation of ligation reactions, and they allow blue/white screening. They also have mutations to maintain plasmid quality and insert stability (e.g., endA–, recA–).
For protein expression, the KRX bacteria has a chromosomal copy of the T7 RNA polymerase gene driven by a rhamnose promoter. The rhamnose promoter is repressed by glucose and highly activated by rhamnose, giving precise control of recombinant protein expression. KRX also contains mutations to minimize proteolysis of over-expressed proteins (ompT– and ompP–), thus improving protein yield and integrity.
For maximum protein expression, inductions are traditionally done overnight. While this can result in higher protein yields, long inductions can have a negative impact on protein activity. Here we examined protein expression and activity at various times after induction in standard Luria broth (LB) and compared activity to rich Terrific Broth (TB).
Effect of Time on Protein Expression Levels and Activity
To examine the effect of induction time on protein expression, the pFN6A (HQ) Flexi® Vector containing the coding region of Renilla (pFN6A-hRluc) or firefly luciferase (pFN6A-luc) was transformed and expression induced in Single Step (KRX) Competent Cells as described in the Single Step (KRX) Competent Cells Technical Bulletin #TB352. Overnight cultures were grown at 37°C in the presence of 0.4% glucose to minimize background protein expression, then diluted 1:100 in 50ml of LB and incubated at 37°C until the cultures reached 0.4–0.5 O.D.600. The cultures were transferred to 25°C and induced using 0.1% rhamnose (final concentration). Aliquots were removed and measured for culture density (O.D.600) at various time points and then analyzed for luciferase activity using the Renilla Luciferase Assay System (Cat.# E2810) or the Bright-Glo™ Luciferase Assay System (Cat.# E2610), or lysed in FastBreak™ Cell Lysis Reagent (Cat.# V8571) for gel analysis.
An increase in Renilla luciferase protein was visible by gel analysis as early as 2 hours after induction (Figure 2). At 4 hours, more active Renilla luciferase was present per O.D.600 of culture than was present after overnight incubation. The culture density continued to increase throughout the incubation (O.D.600 = 2.61 and 5.09 for 4 hours and 20 hours, respectively); therefore, more total Renilla luciferase activity was measured at the later time points, although the activity per O.D.600 was lower. Firefly luciferase activity increased the most within the first 4 hours of induction, with a moderate increase continuing through later time points.
Effect of Media On Protein Expression and Activity
Terrific Broth is a richer medium than LB, resulting in greater cell mass and allowing higher recombinant protein expression levels. The short 4-hour induction and standard overnight induction were compared for protein expression in LB and TB (Figure 3, Panel A). The activity of Renilla luciferase (Figure 3, Panel B) was significantly higher in TB versus LB, which is in direct contrast to firefly luciferase (Figure 3, Panel C). These data emphasize the need to empirically determine optimal expression conditions for each protein.
These data demonstrate that a short 4-hour induction protocol can be used to express significant quantities of active protein from Single Step (KRX) Competent Cells. This allows induction and analysis of recombinant protein expression on the same day. Overnight induction times can be used to generate more total protein, but shorter times can give higher amounts of active protein per O.D. of culture. Optimal induction time and media conditions will depend on the protein expressed and the needs of the researcher. Additional examples of the utility of this short induction protocol are given in the Promega Notes article, “Compatibility of Single Step (KRX) Competent Cells with the MagneGST™ Pull-Down System”.
We wish to acknowledge Mike Slater, Kate Zhao, Jim Hartnett, Sarah Wheeler, and Gary Kobs.