myTXTL Antibody/DS Cell-Free Expression Kit

High-performance cell-free expression of active antibodies and proteins with disulfide bonds

Product Description

The myTXTL Antibody/DS Kit from Daicel Arbor Biosciences is a cutting-edge solution for efficient, high-yield expression of disulfide bond-containing proteins (such as antibodies) as well as non-disulfide bond-containing proteins.

• Rapidly produces complex proteins with and without disulfide bonds
• Single-day workflow
• Skip lysis/purification
• No cloning required, use linear or plasmid DNA
• Maximum versatility

This kit provides industry-leading yields of disulfide bond proteins, even for complex constructs such as Fab and IgG. All without sacrificing the speed and convenience that researchers have come to appreciate from the myTXTL system—simply drop in your linear or plasmid DNA and go!

Each myTXTL Antibody/DS Kit includes the DS Master Mix, the Helper Plasmid (for T7 RNA polymerase expression), and the T7 GLuc Control Plasmid (for T7 promoter-based expression of GLucDura).

If you are interested in expressing only proteins without disulfide bonds, consider the myTXTL Pro Kit instead for even higher protein yields.

Applications

• Antibody screening (ELISA/SPR)
• High-throughput protein or enzyme screening (variant libraries / metagenomics)
• De novo protein design testing
• Enzyme discovery/optimization
• Protein engineering
• Biosensors/gene circuits
• CRISPR-Cas/transposase assays
• Metabolic pathway optimization
• Rebooting bacteriophages
• Teaching labs
• And more!

Performance

Industry-leading performance to express disulfide-bond proteins

myTXTL Antibody/DS Master Mix and three competitor kits marketed for cell-free expression of disulfide-bond containing proteins were used to express four different disulfide-bond containing proteins: 2 Trastuzumab antibody constructs (Fab and IgG), FDA-approved Reteplase, and Gaussia Luciferase (GLucDura). The myTXTL Antibody/DS Master Mix expressed the greatest concentration of all four proteins.

Figure 1. Comparison of protein concentrations following cell-free expression. Expression of active protein was quantified by activity assay alongside a standard for Reteplase, GLucDura, and IgG Trastuzumab. Fab concentration was determined following bead purification.

Rapidly produce active antibody constructs

Antibody constructs representing a VHH, ScFv, Fab, and full IgG were expressed from a T7 promoter for 16 hours using the myTXTL Antibody/DS Kit. Analysis by antigen pull-down and SDS-PAGE visualization was completed less than 24 hours from myTXTL reaction start time. All antibody constructs successfully bound antigen.

Figure 2: SDS-PAGE visualization of antibody: antigen pull-down from dilute myTXTL reactions. Antibodies were expressed in myTXTL Antibody/DS Master Mix and complexed with antigen prior to magnetic bead purification by His-tag (lane 1-2), Twin-Strep-tag™ (lane 3), and Protein A (lane 4). 

Large scale ScFv expression and evaluation

A Trastuzumab ScFv-His construct was expressed for 16 hrs in 400 uL myTXTL Antibody/DS Master Mix and purified with Magnetic NTA beads to yield over 100 μg ScFv. This yield and purity was enough to perform both a multipoint ELISA and SPR Analysis. The KD of 14.5 nM determined for myTXTL-produced ScFv is comparable to the FDA listed KD of 5 nM for Trastuzumab IgG.

Figure 3. Trastuzumab ScFv and HER2 binding analysis by SPR and ELISA. SPR was conducted with immobilized ScFv and 12.5 nM – 200 nM HER2 injections. ELISA performed using HER2coated wells. Data generated by Somnath Mukherjee, University of Chicago.