The Expressway™ Maxi Cell-Free Expression System is designed to perform two hundred 25-µl, five 1-ml, or one 5-ml (initial volume) reactions or one to several large reactions, depending on your needs. This system includes the E.coli slyD- Extract, 2.5X IVPS reaction buffer, feed buffer, 19 amino acid mix, methionine, DNase/RNase-free water, T7 Enzyme Mix, and a positive expression control vector. The Expressway™ Maxi Cell-Free E. coli Expression System with pEXP5-NT/TOPO® and pEXP5-CT/TOPO® also includes the pEXP5-NT/TOPO® and pEXP5-CT/TOPO® TA Expression Kits. Each kit contains the pEXP5-NT/TOPO® or pEXP5-CT/TOPO® vector, other reagents to facilitate TOPO® Cloning, and One Shot® TOP10 Chemically Competent E. coli. Store the vectors at -20°C. Store all other components at -80°C. Guaranteed stable for 6 months when properly stored.

描述

The Expressway™ Maxi Cell-Free E. coli Expression System uses an efficient, coupled transcription/translation reaction to produce milligram quantities of soluble, functionally active protein in 4-6 hours. The procedure can be performed in a single reaction tube and is easily scalable without the need for specialized equipment. The TOPO TA Cloning® expression vectors (5-min cloning with 95% efficiency) are provided for optimal expression results. In addition to all the advantages of an open expression system, Expressway™ Maxi technology provides a means to produce high levels of recombinant protein that may be easily detected and purified for various downstream applications.

Milligram level protein production within 4-6 hours
The key to the Expressway™ Maxi technology is the unique formulation of the Feed Buffer that boosts the reaction productivity so that more than one milligram of protein is produced in a 2-ml reaction within 4-6 hours (Figure 1). The formulation of the lysate enables protein synthesis using either circular or linear templates.

High-throughput (HTP) compatible
This system is also designed for HTP expression. One kit is good for 200 x 50-reactions, which can be accommodated by 2 x 96-well plates. Without feed buffer, such reactions need only two hour’s time. Once a positive expression is detected, scale-up protein production can be performed with the large Expressway™ cell-free format or in E. coli cell-based systems.

Optimized vectors
pEXP5 TOPO® TA vectors are optimized for use in the Expressway™ Milligram system (Figure 2). They are designed to minimize additional amino acid sequences that may interfere with native protein folding and functionality. Features of these vectors include:

• pEXP5-NT/TOPO® expression vector provides N-terminal histidine tag and TEV protease recognition site

• pEXP5-CT/TOPO® expression vector can be used for expressing proteins with a C-terminal histidine tag or for native protein expression by introducing a stop codon at the end of your gene of interest

Simple and fast procedure
The Expressway™ Maxi Cell-Free Expression System provides all the components needed for optimal cell-free protein production. The system includes an E. coli extract, IVPS reaction buffer, Feed Buffer, T7 enzyme mix, 19 amino acid mix, and individual tubes of methionine. To start a 2-ml reaction, mix the reaction buffer, 19 amino acid mix, your choice of labeled or unlabeled methionine and cysteine, T7 enzyme mix, and your DNA template (with T7 promoter) with the E. coli extract. After a 30-minute incubation, add the Feed Buffer. Milligram-levels of active protein will be synthesized within 4-6 hours. To perform HTP expression, simply premix all the component and aliquot into a HTP format.

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