LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells.
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.
Goda
Natsuko
N
Division of Molecular Biophysics, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Tenno
Takeshi
T
Inomata
Kosuke
K
Iwaya
Naoko
N
Sasaki
Yoshiyuki
Y
Shirakawa
Masahiro
M
Hiroaki
Hidekazu
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D002614:Chelating Agents; D002846:Chromatography, Affinity; D005453:Fluorescence; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D028581:Lanthanoid Series Elements / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D011506:Proteins; D011993:Recombinant Fusion Proteins