InTag™ trans-membrane proteins

InTag™ Trans-membrane Proteins

GenTarget’s InTagTM high efficiency protein delivery system lets you directly introduce proteins into cells at 10 to 40 times higher efficiency than a poly-A fusion tag with little to no effect on protein function. 

Advantages of using GenTarget’s InTagTM protein delivery method are: 

  • High efficiency protein delivery–so that much less protein is required for functional assays
  • Able to deliver large proteins (>1000 amino acids)
  • Cell type-independent—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 

The direct introduction of protein into cells is a critical step in studying protein function.  Unlike DNA transfection, in which the transcriptionally active gene is introduced into living cells where the protein is made by cellular machinery, protein delivery does not introduce any exogenous DNA and is independent of cellular transcription and translation machinery. Therefore, it avoids the potential risk of insertional mutagenesis in the host genome and the delivered protein functions immediately upon entering the cell.

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 unequivocally the easiest for use with  non-cytotoxic proteins and is applicable for large scale applications; it is, therefore, the most attractive therapeutic approach. 

Protein fusion delivery depends upon short cationic peptides known as protein transduction domains (PTDs) that are fusioned to target proteins.  PTDs, which generally contain multiple (7 ~15) arginines can efficiently cross the plasma membrane by a mechanism that is incompletely understood.

Native PTDs  found in many viral proteins demonstrate the ability to transit the plasma membrane.  Among these are TAT protein from HIV-1, influenza virus hemagglutinin protein (HA2), 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 poly-Arginine fusion tag, depending upon cell type. Target proteins fusioned with the InTagTM PTD are efficiently delivered into a variety of living cells. 

Gentarget’s InTagTM protein delivery domain is incorporated into our Eco PCR cloning products (Cat#:  to be launched) 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. 

GenTarget also provides many purified, transfection-ready proteins (see product links). 

References

  • Yohei MUKAI, Biol. Pharm Bull, 29(8):1570-1574, 2006;
  • Panagiotis S. Kabouridis, TRENDS in Biotechnology Vol.21 No.11 November 2003;
  • Alan Ho, et al., CANCER RESEARCH 61, 474-477, January 15, 2001;