Thermo Fisher Scientific Expressway Maxi Cell-Free E. coli Expression System

Invitrogen™ Expressway™ Maxi Cell-Free E. coli Expression System

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. Expressway™ Maxi technology enables high levels of recombinant protein production that can be easily detected and purified for 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 reaction productivity, allowing more than one milligram of protein to be produced in a 2‑ml reaction within 4–6 hours. The lysate formulation supports protein synthesis using either circular or linear templates.

High-Throughput (HTP) Compatible

This system is designed for HTP expression. One kit supports 200 × 50‑µl reactions, suitable for 2 × 96‑well plates. Without Feed Buffer, reactions complete in just two hours. Once expression is confirmed, scale-up production can be performed using the large Expressway™ cell-free format or E. coli cell-based systems.

Optimized Vectors

pEXP5 TOPO™ TA vectors are optimized for use in the Expressway™ Milligram system. They minimize additional amino acid sequences that may interfere with native protein folding and functionality.

• pEXP5‑NT/TOPO™: Provides N-terminal histidine tag and TEV protease recognition site
• pEXP5‑CT/TOPO™: Enables C-terminal histidine tag expression or native protein expression by introducing a stop codon at the end of the gene of interest

Simple and Fast Procedure

The Expressway™ Maxi Cell-Free Expression System provides all components needed for optimal cell-free protein production, including E. coli extract, IVPS reaction buffer, Feed Buffer, T7 enzyme mix, 19 amino acid mix, and methionine tubes.
To start a 2‑ml reaction:

1. Mix reaction buffer, amino acids, labeled/unlabeled methionine and cysteine, T7 enzyme mix, and DNA template (with T7 promoter) with the E. coli extract.
2. Incubate for 30 minutes.
3. Add Feed Buffer.
   Milligram-levels of active protein are synthesized within 4–6 hours.
   For HTP expression, premix all components and aliquot into HTP format.

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