High-level protein expression without the need to monitor cell growth

Overnight Express Autoinduction System 2 provides a complete, chemically defined medium for high-level protein expression with the pET System and other IPTG-inducible expression systems without the need to monitor cell growth. All system components have a known chemical composition, resulting in consistent product performance and the elimination of lot-to-lot variability. System 2 also features the capability to label proteins with selenomethionine (Se-Met) for downstream crystallization and x-ray diffraction studies.

The system includes six concentrated sterile solutions: OnEx™ solutions 1 through 6. OnEx Solutions 1, 2, and 3 are the same components included in the Overnight Express System 1. OnEx Solution 4 provides trace metals to minimize growth limitations associated with mineral deficiencies and satisfy the metal requirements of metal-containing target proteins even at high expression levels. OnEx Solution 5 is a mixture of amino acids lacking methionine. OnEx Solution 6 is a separate methionine solution. Sufficient methionine (Met) is provided to support growth of the Met auxotroph B834 while providing the ability to reduce the level of unlabeled Met for selenomethionine incorporation by Met auxotrophs.

The Overnight Express Autoinduction System 2 is extremely convenient for routine expression of proteins in multiple cultures and is ideal for high-throughput parallel analysis of protein expression, solubility screening, and purification from multiple expression clones. The tedium of preparing a defined medium from dozens of components has been simplified with the ready-to-use sterile solutions included in the Overnight Express Autoinduction System 2.

The Overnight Express™ Autoinduction Systems enable regulated protein expression in E. coli without monitoring the culture or adding inducer during cell growth. These unique culture media are based on technology by F. William Studier at Brookhaven National Laboratory. The products include Overnight Express Instant TB Medium, System 1, and System 2. 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 Autoinduction Systems feature high-level protein production in the pET and other IPTGinducible bacterial expression systems without the need to monitor cell growth. Cell mass and target protein yield are often increased severalfold as compared with conventional protocols using induction with IPTG.



Overnight Express Instant TB Medium
Overnight Express Instant TB Medium is a complete granulated culture medium for high-level protein production in the pET and other IPTG-inducible bacterial expression systems without the need to monitor cell growth. Cell mass and target protein yield are often increased several-fold as compared with conventional protocols using induction with IPTG. 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. The Overnight Express Instant TB Medium is extremely convenient for routine expression of proteins in multiple cultures and is ideal for high-throughput parallel analysis of protein expression, solubility, and purification from multiple expression clones.



Overnight Express Instant TB Medium is supplied in two formats. EasyPak is an aluminum foil pouch containing 60 g granulated medium sufficient for 1 L culture. Just add the EasyPak contents to 1 L water, supplement with 10 ml glycerol, and microwave for 2 minutes to sterilize. In addition, the Overnight Express Instant TB Medium is supplied in 1 kg bottles. The granules ensure rapid and uniform dissolution in water, and prevent clumping of the medium and inhalation of the airborne powder.



Overnight Express Autoinduction System 1
With Overnight Express™ Autoinduction System 1, a period of cell growth is followed by spontaneous induction of protein expression–without monitoring cell density and without conventional induction with IPTG. 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.

The kit contains three components, OnEx™ Solutions 1, 2, and 3. OnEx Solution 1 is a blend of carbon sources optimized for tightly regulated uninduced growth to high cell density, followed by high-level induction. OnEx Solution 2 is a concentrated buffer and nitrogen-source blend that mediates metabolic acid production and provides additional nitrogen for increased protein synthesis. OnEx Solution 3 provides magnesium for maximal cell density. Addition of these components to traditional glucose-free E. coli culture media, such as LB broth or TB media results in high cell densities, autoinduction of expression, and maximum soluble protein yields (Grabski, et al 2003).

The Overnight Express Autoinduction System is extremely convenient for routine expression of proteins in multiple cultures and is ideal for high-throughput parallel analysis of protein expression, solubility, and purification from multiple expression clones. The Overnight Express Autoinduction System together with the Novagen RoboPop™ Solubility Screening Kit and RoboPop Purification Kits allow rapid optimization of host-vector combinations, expression conditions, and purification parameters for high-throughput production of proteins for structural or functional analysis.



Overnight Express Autoinduction System 2
Overnight Express™ Autoinduction System 2 provides a complete, chemically defined medium for high-level protein expression with the pET System, and other IPTG-inducible expression systems, without the need to monitor cell growth. All system components have a known chemical composition, resulting in consistent product performance and the elimination of lot-to-lot variability. System 2 also features the capability to label proteins with selenomethionine (Se-Met) for downstream crystallization and x-ray diffraction studies.

The system includes six concentrated sterile solutions: OnEx™ solutions 1 through 6. OnEx Solutions 1, 2, and 3 are the same components included in the Overnight Express System 1 (see preceding page). OnEx Solution 4 provides trace metals to minimize growth limitations associated with mineral deficiencies and satisfy the metal requirements of metal-containing target proteins, even at high expression levels. OnEx Solution 5 is a mixture of amino acids lacking methionine. OnEx Solution 6 is a separate methionine solution. Sufficient methionine (Met) is provided to support growth of the Met auxotroph B834 while providing the ability to reduce the level of unlabeled Met for selenomethionine incorporation by Met auxotrophs.

The Overnight Express Autoinduction System 2 is extremely convenient for routine expression of proteins in multiple cultures and is ideal for high-throughput parallel analysis of protein expression, solubility screening, and purification from multiple expression clones. The tedium of preparing a defined medium from dozens of components has been simplified with the ready-to-use sterile solutions included in the Overnight Express Autoinduction System 2.

Overnight Express Autoinduction System 1

Two Overnight Express™ Autoinduction Systems (System 1 and System 2) are available, both featuring high-level protein production in the pET and other IPTG-inducible bacterial expression systems without the need to monitor cell growth. Cell mass and target protein yield are often increased several-fold as compared with conventional protocols using induction with IPTG. Also available is Overnight Express Instant TB Medium.

Features and Benefits:

• High cell densities and protein expression levels
• Complete chemically defined medium; minimal lot-to-lot variability
• No need to monitor cell growth rate or add inducer
• Ideal for Se-Met labeling of proteins to be crystallized for x-ray diffraction studies
• Ideal for pET Expression System or other IPTG-inducible bacterial systems
• Induction of numerous expression clones simultaneously
• Compatible with cultures grown in flasks, culture tubes, and deep-well plates
• Minimal sample handling

References:
Grabski, A., et al. 2003. inNovations 17, 3.

Components:

•	20 ml or 100 ml	OnEx™ Solution 1
•	50 ml or 125 ml	OnEx Solution 2
•	1 ml	OnEx Solution 3
•	1 ml	OnEx Solution 4
•	20 ml or 100 ml	OnEx Solution 5
•	20 ml or 100 ml	OnEx Solution 6