LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells

Abstract

Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.

Cellular Delivery of Noncovalently-Associated Macromolecules by Cell- Penetrating Peptides

“…Using a phage display system [85] or a random peptide library [86], synthetic CPPs have been systematically created and screened. Furthermore, CPP could serve as a useful tag to facilitate unidirectional cloning of PCR-amplified DNA fragments in addition to cellular protein delivery [87]. These properties emphasize the versatility of CPPs.…”

A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR

“…The PRESAT-vector method is designed for unidirectional TA cloning that facilitates the construction of fusion protein expression vectors with limited effort and low background self-ligation. We developed several PRESAT vectors with many different linkers suited for different purposes [ 41 , 43 , 44 ]. All the vectors contained the PRESAT linker, which was designed for unidirectional TA cloning of PCR-amplified DNA fragments.…”

The Best and the Brightest: Exploiting Tryptophan-Sensitized Tb3+ Luminescence to Engineer Lanthanide-Binding Tags

“…LBTs have been used for luminescence-based visualization on polyacrylamide gels [ 13 , 28 ], as magnetic fi eld paramagnetic alignment agents in protein NMR experiments [ 29 -31 ], in fl uorescence microscopy [ 32 ] and as partners in luminescence resonance energy transfer (LRET) studies [ 33 ]. In addition, solid-phase peptide synthesis has been used for the development of LBTs containing unnatural amino acids in place of tryptophan at position 7, with side chains that can be excited at lower-energy wavelengths and sensitize Eu 3+ [ 34 ].…”