Engineered protein transmembrane domain (PTD)
The direct introduction of protein into cells is a critical step in studying protein function. Unlike DNA transfection, in which the transcriptionally active DNA/gene is introduced into living cells where protein is made by cellular machinery, protein delivery does not introduce any exogenous DNA and is independent of the cellular transcription and translation machinery. It thereby avoids the potential risk of insertional mutagenesis in the host genome and the delivered protein functions immediately upon entry into the cells.
Several methods for protein delivery have been developed, including electroporation, microinjection, protein fusion, and traditional cationic lipid carriers. Among these methods, protein fusion delivery is no doubt the easiest for use with non-cytotoxic proteins and is applicable for large-scale applications, making it a more attractive therapeutic approach.
Protein fusion delivery depends upon a protein transduction domain (PTD) that is fusioned with the target protein domain. PTDs are short cationic peptides that can efficiently cross the plasma membrane. The mechanism for PTD entry into cells is incompletely understood. A basic form of PTD is multiple (7 ~15) arginines.
The native PTDs found in many viral proteins demonstrate membrane transiting; among these are TAT protein from HIV-1, influenza virus hemagglutinin protein (HA2), the herpes simplex virus 1 (HSV-1) DNA-binding protein VP22 and Drosophila Antennapedia (Antp) homeotic transcription factor.
From phage screening and point mutation screening results, GenTarget has developed InTagTM, a proprietary, high efficiency protein delivery system utilizing an engineered PTD. The 37 amino acid InTagTM PTD shows little to no effect on target protein function, and demonstrates greater than 10- to 40-fold higher efficiency than a ploy-arginine fusion tag, depending upon cell type. Target proteins fusioned with the InTagTM PTD are efficiently delivered into a variety of living cells.
The advantages of using Gentarget’s InTagTM protein delivery method are:
- High efficiency protein delivery–so that much less protein is required for functional assays
- Ability to deliver large proteins (>1000 amino acids)
- Cell type independence—enabling use with many different cultured mammalian cells
- Rapid delivery for readily acquired protein function (<5min)
- Reversible delivery of protein function
- Non-invasive delivery—simply add into cell culture
- Controllable delivery—by varying the concentration
- Targeted delivery when fusioned with a targeting protein
GenTarget’s InTagTM protein delivery domain was incorporated into the target expression vectors for E Coli or mammalian expression of transfection-ready proteins. Following purification, proteins expressed with the InTagTM PTD, can simply be applied to cultured mammalian cells for in vitro functional assay.
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