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Overnight Express™ Autoinduction Technology  The Overnight Express™ Autoinduction Systems are designed for high-level protein expression with the pET and other IPTG-inducible bacterial expression system without monitoring cell growth. The problems associated with inadvertent expression of target proteins and attempts to understand effects of complex and defined media components on growth, catabolite repression, and induction of T7 expression strains led to the formulation of media to support growth to saturation without induction as well as fully defined and complex autoinduction media1. In autoinduction media, cultures reliably grow uninduced to relatively high density and then spontaneously induce high levels of target protein without the need to monitor cell density or add IPTG.
Key components of the autoinduction formulations are now available in the Overnight Express Autoinduction System 1, Overnight Express Autoinduction System 2 , and Overnight Express Instant TB Medium.  R&D 100 Award
F. William Studier, a biophysicist at the U.S. Department of Energy's Brookhaven National Laboratory, has won a 2004 R&D 100 award for developing a new process that simplifies the production of proteins in the widely used T7 gene expression system. Through a long-term successful scientific and business relationship with Brookhaven National Laboratory, EMD Biosciences was granted exclusive licensing rights for this technology, and has made it commercially available as the Overnight Express™ Autoinduction System. The new method simplifies the production of many proteins in parallel and is useful for biomedical research or for industrial production of proteins to use as enzymes, diagnostics, vaccines, therapeutics and targets for developing pharmaceuticals. R&D 100 Awards are given annually by R&D Magazine to the top 100 technological achievements of the year. Typically, these are innovations that transform basic science into useful products. The awards were presented in Chicago on October 14.
The 2004 R&D 100 award-winning Overnight Express autoinduction system facilitates the basic science of protein expression. The method is based on media components that are metabolized differentially to promote growth to high density and automatically induce protein expression from lac promoters. Cell mass and target protein yield are often increased several-fold as compared with conventional protocols using induction with IPTG.
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New Overnight Express
Instant TB Medium | | New, easy-to-use granulated format—just add water!  Protein expression has never been as easy as with Overnight Express Instant TB Medium, a complete, granulated culture medium, which combines Overnight Express System 1 and Terrific Broth (TB). The medium is available in two package types: EasyPak pouches and 1-kg bottles.The EasyPak foil pouch contains 60 g granulated medium. Just add the EasyPak contents to 1 L of water, supplement with 10 ml glycerol, and either autoclave or microwave to sterilize. The granules ensure rapid and uniform dissolution in water, prevent aggregation of the medium, and reduce the generation of airborne particulates.
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Overnight Express
Autoinduction System 1 | | | SDS-PAGE analysis of crude and purified proteins from cultures induced using Overnight ExpressSystem 1 or IPTG
pET recombinants encoding b-gal, NusA, and GST fusion proteins were transformed into BL21(DE3) cells. Cells were cultured and protein expression was induced in parallel cultures either by Overnight Express System 1 autoinduction (OE1) or 1 mM IPTG (IPTG). Following induction, cells were harvested by centrifugation and extracted with BugBuster® Master Mix to produce a crude extract. Equal volumes (7 μl) of the crude protein extract (C) were analyzed by SDS-PAGE (10–20% gradient gel) and Coomassie blue staining. The remainder of the extract was used for robotic affinity purification using Ni-NTA His•Bind® Resin. Samples (2 μg) of the purified fraction (P) were analyzed by SDS PAGE on the same gel. |
The Overnight Express™ Autoinduction Systems enable regulated protein expression in E. coli without monitoring the culture or adding inducer during cell growth. The products include Overnight Express System 1, System 2, and Instant TB Medium. The granulated Instant TB Medium formulation combines Overnight Express System 1 and Terrific Broth (TB) into a rich culture medium, making autoinduction of protein expression even more convenient. The Overnight Express System 1 contains three solutions. OnEx™ Solution 1 is a blend of carbon sources optimized for tightly regulated, uninduced growth to high cell density, followed by induction with lactose and continued growth. OnEx Solution 2 is a concentrated buffer that maintains pH throughout metabolic acid production and supplies the additional nitrogen necessary to support increased protein synthesis. OnEx Solution 3 provides critical magnesium for maximum cell density. Addition of these Overnight Express components to glucose-free complete media, such as Luria-Bertani (LB) broth or TB (5), allows autoinduction, high cell densities, and increased yields of soluble target proteins with pET (2) or other IPTG-inducible systems. Expression levels can be further increased by careful host selection and optimizing growth temperature and harvest time. Because lactose is used for induction, the expression hosts must produce functional lactose permease (encoded by the lacY gene) and b-galactosidase (b-gal, encoded by the lacZ gene). Addition of kit components to traditional E. coli culture media such as LB, TB, or animal-free Veggie™ Media results in high cell densities, auto-induction of expression, and maximum soluble protein yields. The Overnight Express Autoinduction System is extremely convenient for routine expression of proteins in multiple cultures and is ideal for high-throughput parallel analys is of protein expression, solubility and purification from multiple expression clones. The kit provides sufficient reagents for autoinduction from up to 1 L or 5 L of media.
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Overnight Express
Autoinduction System 2 | | |
Mass spectroscopy analysis demonstrates that the target protein produced with Overnight Express System 2 autoinduction incorporated Se-Met. The target protein shown has 12 methionine residues and the MS data shows an average of 10.4 substitutions, or 87% incorporation. Spectra provided by the Mass Spectrometry/Bioanalytical Facility at the University of Wisconsin Biotechnology Center. |
The Overnight Express System 2 contains OnEx™ Solutions 1–3 plus three additional components, OnEx Solutions 4–6. OnEx Solution 4 provides trace metals below toxic levels to minimize growth limitations associated with mineral deficiencies and saturates almost any metal-containing target protein even at high expression levels. OnEx Solution 5 is an amino acid mixture lacking methionine, cysteine, and tyrosine. OnEx Solution 6 is an individual solution of methionine (Met). Adding these six components to sterile water results in a defined medium capable of promoting high cell densities, enabling autoinduction of expression, producing maximum soluble protein yields, and, if desired, efficient labeling of target proteins by addition of selenomethionine (Se-Met).
View product details and ordering information | | | | Reference | - F. W. Studier (personal communication)
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