What is autoinduction?
Autoinduction refers to bacterial cultures grown in media containing specific components that, after an initial period of tightly-regulated, uninduced growth, automatically induce target protein expression, without IPTG. Typically,autoinduced expression produces a greater proportion of soluble target protein than does IPTG-induced expression. Overnight Express™ Autoinduction Systems use optimized media components to promote culture growth to high cell densities, followed by lactose-induced protein expression from lac-based promoters. This method provides higher protein yields and greater convenience compared to standard IPTG induction. What specific media components are usedin the Overnight Express Autoinduction Systems?
The Overnight Express Autoinduction Systems use an
optimized blend of carbon sources, including lactose.
The carbon sources are metabolized differentially to initially promote tightly regulated, uninduced growth, followed by autoinduction with lactose and continued cell growth, which results in high cell density at harvest. OnEx™ Solution 1 provides the blend of carbon sources.
To grow bacterial cultures to high cell densities,
metabolic acids must be buffered to maintain pH near
neutral. For increased protein expression, additional nitrogen is also necessary. OnEx Solution 2 provides both a concentrated buffer and an additional nitrogen source, so that cultures attain high cell densities and increased protein expression, even when grown in shake flasks or 96-well plates.
Magnesium is essential for cell growth, and higher
concentrations of magnesium have been shown to
increase the viability of cultures stored at 4˚C for
several weeks (1). OnEx Solution 3 provides the critical
magnesium necessary to attain maximum cell density
and improved culture viability. What are the differences between the three Overnight Express Products?
The Overnight Express Autoinduction System 1 (2) includes OnEx Solutions 1-3, which are ready to be added to any glucose-free complex medium, such as Luria-Bertani (LB) broth or Terrific Broth (TB). The Overnight Express Autoinduction System 2 includes OnEx Solutions 1-3 and OnEx Solutions 4-6, which together comprise a complete, defined autoinduction medium when added to water. Using the System 2 defined autoinduction medium, target proteins can be efficiently labeled with selenomethionine (Se-Met). The Overnight Express Instant TB Medium is a complete, granulated, autoinduction medium, which includes the components found in the OnEx Solutions 1-3. Simply add the granules to water, supplement with glycerol, microwave or autoclave to sterilize, and inoculate. Should IPTG be added at any point during culture growth?
No. This method is based on media components that are metabolized differentially to promote growth to high density and automatically induce protein expression. OnEx™ Solution 1 is the induction solution, that is a blend of carbon sources to permit high-density cell growth in the absence of expression followed by induction at high cell density. Are the Overnight Express Autoinduction Systems compatible with any expression host?
Because lactose is used for induction, expression hosts producing functional Lac permease (encoded by the lacY gene) and b-galactosidase (encoded by the lacZ gene) are required. Strains with nonfunctional Lac permease will not efficiently transport lactose for induction, and strains with nonfunctional b-galactosidase will not convert a portion of the transported lactose to the necessary allolactose inducer. The system is compatible with host strains such as BL21, Rosetta™, Rosetta 2, and their DE3 lysogen derivatives. If the expression vector uses a T7lac promoter, as many of the pET vectors do, a host strain without a pLysS plasmid is recommended. The combination of the T7 lysozyme (expressed by the pLysS plasmid) and the lac repressor (encoded by vectors carrying the T7lac promoter) causes significantly reduced protein expression in the Overnight Express Autoinduction Systems. When the “plain” T7 promoter is used, the low level of lysozyme produced by pLysS has little effect on expression of target proteins. In what bacterial protein expression system has the Overnight Express™ Autoinduction Systems been tested?
Overnight Express Autoinduction Systems have been tested primarily with the pET protein expression system (we have extensively tested T7lac promoter vectors). However, experiments with other IPTG-inducible promoters (i.e., tac and trc promoters) have also been successful. Based on the overall mechanism of action of the system, Overnight Express should be compatible with any IPTG-inducible promoter used for protein expression.  |
Plasmids were transformed into BL21(DE3) (pET43.1) or BL21 (non-T7 promoters), and grown at 37°C in 30 ml LB + carbenicillin (lane 1) or LB + carbenicillin plus OnEx™ Solutions 1, 2, and 3 (lanes 2, 3, 4, and 5). The culture in lane 1 was induced with 1 mM IPTG at an OD600 of 0.6 and harvested after 3 h. Other cultures were autoinduced overnight (16 h). M: Perfect Protein™ Markers, 10–225; 1: pET43.1 (T7lac, 66.1 kDa) IPTG; 2: pET43.1 (T7lac, 66.1 kDa); 3: pGEX (tac, 24.5 kDa); 4: pMal (tac, 43 kDa); 5: pTrc (trc, 37 kDa; the insert in pTrc is vTPA, which contains 32 rare codons) |
Does Overnight Express work in fermentation systems?
Yes, Overnight Express was tested in a 15-L fermentation system and produced higher cell densities and better protein yields per liter than the IPTG-induced system to which it was compared. 
How long does it take before induction begins? How long should I grow the cultures? What will the cell density be when my cultures reach saturation?
With the Overnight Express system, cultures grow to relatively high densities and then spontaneously induce high levels of target protein. When inducing with IPTG, all of the cell's energy is devoted to producing target protein, while with the Overnight Express systems the cells produce target protein and continue to grow. This gradual induction leads to higher cell densities, which can lead to increased expression levels. Therefore, it is important to grow the cells to stationary phase when using the Overnight Express Systems. When using the cell culture guidelines outlined in the User Protocol and growing the cells at 37°C, stationary phase is usually reached as quickly as 8–10 hours. When lower incubation temperatures are used, saturation may only be reached by incubating for 24 hours or more. Continued incubation for several hours after stationary phase appears to have no deleterious effects. The cell density at harvest (OD600) will vary from protein to protein. If it is not known if the cultures have reached saturation, we recommend measuring OD 600 and continuing to grow the cells for a couple of more hours. If the absorbance has not changed in this time frame, the culture has reached saturation. Growth and induction at 25°C or 30°C may be optimal if you want to export the target using the signal sequence leaders present in a number of pET vectors or improve the yield of soluble protein. When lower incubation temperatures are used, saturation may only be reached by incubation for 24 hours or more. When using lower temperatures it can be helpful to first incubate the culture at 37°C for 3–4 hours and then place the culture at room temperature until the culture reaches stationary phase (at least overnight). Can I use Overnight Express for the expression of proteins that are toxic to E. coli ? Results will vary from protein to protein, however, in general, the Overnight Express Systems would be suitable for potentially toxic proteins. Because basal expression is tightly regulated and the induction is gradual, the Overnight Express Systems may help to improve the expression of some toxic proteins. Expression of several potentially toxic mammalian proteins has been successful with Overnight Express. | | |
pET recombinants encoding the indicated His•Tag® fusion proteins were transformed into BL21(DE3). Cultures were grown and induced as described in the Table, and target proteins were extracted and purified using the RoboPop™ Ni-NTA His•Bind® Purification Kit. Samples (Annexin I: 4 µl; Tubulin alpha-4: 4 µl; Annexin II: + IPTG: 8 µl, Annexin II + OE: 4.5 µl) were analyzed by SDS-PAGE (10–20% gradient gel) and Coomassie™ blue staining. IPTG: IPTG induction; OE: Overnight Express autoinduction; M: Perfect Protein™ Markers, 15–150 kDa |
| | Protein | Cell Density at Harvest1 | Pure Protein Yield2 | | OE3 | IPTG4 | OE | IPTG | | Annexin I | 6.8 | 5.8 | 240 | 248 | | Annexin II | 5.4 | 4.9 | 323 | 165 | | Tubulin alpha-4 | 10 | 8.6 | 45 | 15 | - OD600
- Target proteins were extracted with PopCulture® Reagent and robotically purified according to our user protocol. Pure protein yield was quantified as µg/ml culture (BCA Protein Assay Kit).
- Overnight Express induction was accomplished by inoculating a single colony into 5 ml medium in 10-ml × 24-well plates and incubating overnight (approximately 16 h) at 30°C with shaking at 250 rpm.
- IPTG induction was accomplished by inoculating a single colony into 5 ml medium in 10-ml × 24-well plates and incubating at 16°C with shaking at 250 rpm to an average OD600 of 1.0 followed by addition of IPTG to 1 mM final concentration and incubating an additional 16 h prior to harvest.
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