Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines

被引:16
|
作者
Kita, Ayami [1 ]
Hino, Nobumasa [1 ]
Higashi, Sakiko [1 ]
Hirota, Kohji [1 ]
Narumi, Ryohei [2 ]
Adachi, Jun [2 ]
Takafuji, Kazuaki [3 ]
Ishimoto, Kenji [1 ]
Okada, Yoshiaki [1 ]
Sakamoto, Kensaku [4 ,5 ]
Tomonaga, Takeshi [2 ]
Takashima, Seiji [3 ,6 ,7 ]
Mizuguchi, Hiroyuki [8 ]
Doi, Takefumi [1 ]
机构
[1] Osaka Univ, Grad Sch Pharmaceut Sci, Lab Mol Med, 1-6 Yamadaoka, Suita, Osaka 5650871, Japan
[2] Natl Inst Biomed Innovat Hlth & Nutr, Lab Proteome Res, 7-6-8 Saito Asagi, Osaka 5670085, Japan
[3] Osaka Univ, Grad Sch Med, Ctr Med Res & Educ, 2-2 Yamadaoka, Suita, Osaka 5650871, Japan
[4] RIKEN Ctr Life Sci Technol, Div Struct & Synthet Biol, 1-7-22 Suehiro Cho, Yokohama, Kanagawa 2300045, Japan
[5] RIKEN Ctr Life Sci Technol, Mol Network Control Project, 1-7-22 Suehiro Cho, Yokohama, Kanagawa 2300045, Japan
[6] Osaka Univ, Grad Sch Med, Dept Med Biochem, 2-2 Yamadaoka, Suita, Osaka 5650871, Japan
[7] Japan Sci & Technol Agcy, Core Res Evolut Sci & Technol, Kawaguchi, Saitama 3320012, Japan
[8] Osaka Univ, Grad Sch Pharmaceut Sci, Lab Biochem & Mol Biol, 1-6 Yamadaoka, Suita, Osaka 5650871, Japan
来源
SCIENTIFIC REPORTS | 2016年 / 6卷
关键词
SITE-SPECIFIC INCORPORATION; MAMMALIAN-CELLS; STEM-CELLS; EFFICIENT; GENE; CONSTRUCTION; MUTAGENESIS; SYSTEM; LYSINE;
D O I
10.1038/srep36946
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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, N-epsilon-(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.
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页数:9
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