Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines.
The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Ayami
A
Kita
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nobumasa
N
Hino
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Sakiko
S
Higashi
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kohji
K
Hirota
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Ryohei
R
Narumi
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Jun
J
Adachi
Center for Medical Research and Education, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kazuaki
K
Takafuji
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kenji
K
Ishimoto
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Yoshiaki
Y
Okada
Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.|Molecular Network Control Project, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Kensaku
K
Sakamoto
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Takeshi
T
Tomonaga
Center for Medical Research and Education, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.|Department of Medical Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.|Japan Science and Technology Agency-Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Saitama 332-0012, Japan.
Seiji
S
Takashima
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Hiroyuki
H
Mizuguchi
Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Takefumi
T
Doi
D000072283:A549 Cells; D000256:Adenoviridae / Q000235:genetics*; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry; D005815:Genetic Code; D005822:Genetic Vectors; D057809:HEK293 Cells; D019073:HT29 Cells; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry
D000596:Amino Acids; D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; C573114:N(epsilon)-(benzyloxycarbonyl)lysine; D000604:Amino Acyl-tRNA Synthetases; D008239:Lysine