CloverDirect ~非天然アミノ酸導入用試薬

概要

CloverDirect tRNA Reagents for Site-Directed Protein Functionalizationは、無細胞翻訳系を利用してタンパク質の指定した部位に非天然アミノ酸を導入するための試薬です。蛍光基、ビオチン、PEG鎖、架橋剤を側鎖に持つ非天然アミノ酸や、翻訳後修飾アミノ酸などが用意されています。終止コドンの1つであるアンバーコドンまたは4塩基コドンを組み込んだ遺伝子と本製品を無細胞翻訳系に加えるだけで、非天然アミノ酸導入タンパク質を短時間で効率よく合成することができます。

詳細は取扱説明書製品パンフレットにてご確認ください
CloverDirect1

製品リスト

コード 非天然アミノ酸 コドン 導入可能部位*1 容量
(µL翻訳
反応)
価格
(税抜)
N末端
領域
内部、
C末端
CLD02 TAMRA-X-AF
[5(6)-TAMRA–X : Abs/Em = 546/575nm]
TAMRA-X-AF
amber × 300 ¥28,500
CLD06 5 X 300 ¥128,500
CLD1006 CGGG 300 ¥28,500
CLD2006 5 X 300 ¥128,500
CLD1001 CR110-X-AF
[5-CR110-X : Abs/Em = 498/521nm]
CR110-X-AF
amber × 300 ¥28,500
CLD2001 5 X 300 ¥128,500
CLD2002 CGGG 5 X 300 ¥128,500
CLD01 HiLyte Fluor 488-AF
[HiLyte FluorTM 488 : Abs/Em = 497/525nm]
構造は非公開
amber × 300 ¥28,500
CLD05 5 X 300 ¥128,500
CLD2004 CGGG 5 X 300 ¥128,500
CLD03 ATTO633-AF
[ATTO633-X : Abs/Em = 629/657nm]
構造は非公開
amber × 300 ¥40,000
CLD07 5 X 300 ¥180,000
CLD2008 CGGG 5 X 300 ¥180,000
CLD1009 ATTO655-X-AF
[ATTO655-X : Abs/Em = 663/684nm]
構造は非公開
amber × 300 ¥40,000
CLD2009 5 X 300 ¥180,000
CLD1010 CGGG 300 ¥40,000
CLD2010 5 X 300 ¥180,000
CLD2101 Biotin-AF
[Biotin]
Biotin-AF
amber 5 X 300 ¥185,000
CLD2102 CGGG 5 X 300 ¥185,000
CLD2103 Biotin-X-AF
[Biotin-X]
Biotin-X-AF
amber 5 X 300 ¥185,000
CLD2104 CGGG 5 X 300 ¥185,000
CLD04 Biotin-XX-AF
[Biotin-XX]
Biotin-XX-AF
amber 300 ¥20,000
CLD08 5 X 300 ¥90,000
CLD2106 CGGG 5 X 300 ¥90,000
CLD2203 Lys(Me)
[ε-methyl-Lys]
Lys(Me)
amber 5 X 300 ¥320,000
CLD2204 CGGG 5 X 300 ¥320,000
CLD2205 Lys(Me2)
[ε-dimethyl-Lys]
Lys(Me)2
amber 5 X 300 ¥280,000
CLD2206 CGGG 5 X 300 ¥280,000
CLD2207 Lys(Ac)
[ε-acetyl-Lys]
Lys(Ac)
amber 5 X 300 ¥280,000
CLD2208 CGGG 5 X 300 ¥280,000
CLD2301 PEG4-AF
[Methyl-PEG4]
PEG4-AF
amber 5 X 300 ¥230,000
CLD2302 CGGG 5 X 300 ¥230,000
CLD2303 PEG8-AF
[Methyl-PEG8]
PEG8-AF
amber 5 X 300 ¥230,000
CLD2304 CGGG 5 X 300 ¥230,000
CLD2305 PEG12-AF
[Methyl-PEG12]
PEG12-AF
amber 5 X 300 ¥230,000
CLD2306 CGGG 5 X 300 ¥230,000
CLD2321 BPA
[p-benzoyl-phenylalanine]
BPA
amber 5 X 300 ¥198,000
CLD2322 CGGG 5 X 300 ¥198,000
CLD2323 AcPhe
[p-acetyl-phenylalanine]
AcPhe
amber 5 X 300 ¥198,000
CLD2324 CGGG 5 X 300 ¥198,000
CLD2331 AzoAla
[p-phenylazophenyl-alanine]
azoAla
amber 5 X 300 ¥330,000
CLD2332 CGGG 5 X 300 ¥330,000

※1 : 導入可能部位
非天然アミノ酸の種類によって、導入可能な部位が異なります。
○ : 導入可能
△ : 部位によっては導入が可能
× : 導入は困難

参考文献

1) FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids
Daisuke Kajihara, Ryoji Abe, Issei Iijima, Chie Komiyama, Masahiko Sisido, Takahiro Hohsaka
Nature Methods, 3, 923-929 (2006).

2) Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system
Takayoshi Watanabe, Norihito Muranaka, Issei Iijima, and Takahiro Hohsaka
Biochem. Biophys. Res. Commun., 361, 794-799 (2007).

3) Comprehensive screening of amber suppressor tRNAs suitable for incorporation of non-natural amino acids in a cell-free translation system
Hikaru Taira, Yosuke Matsushita, Kenji Kojima, Kaori Shiraga, Takahiro Hohsaka
Biochem. Biophys. Res. Commun., 374, 304-308 (2008).

4) Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems
Takahiro Hohsaka, Daisuke Kajihara, Yuki Ashizuka, Hiroshi Murakami, and Masahiko Sisido
J. Am. Chem. Soc., 121, 34-40 (1999).

5) 無細胞翻訳系における非天然アミノ酸の導入技術の開発とその応用
芳坂 貴弘, 生化学, 72(3), 247-253 (2007).

6) 遺伝暗号を拡張した人工タンパク質合成系の開発と応用
芳坂 貴弘, 生物物理, 47(2), 124-128 (2007).

アプリケーションデータ

ピンポイント標識タンパク質の発現例(大腸菌無細胞翻訳系)

CloverDirect2

2UAG:
開始AUG直後にUAGコドンを挿入した遺伝子
ProX tag※1 :
ProX tag(UAG)を付加した遺伝子
ゲルへのアプライ量 :
翻訳反応液 0.25μL相当量
蛍光イメージャー検出波長 (上図)
HiLyteFluor488
励起488nm / 検出520 nm
TAMRA
励起532nm / 検出580 nm
ATTO633
励起635nm / 検出670 nm
ウエスタンブロット(下図) :
抗His tag抗体を使用
(ビオチン標識タンパク質の検出には抗ビオチン抗体を使用)

製品使用例

1) A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates.
Nakata H, Ohtsuki T, Sisido M.
Anal Biochem. 2009 Jul 15;390(2):121-5.

2) “Quenchbodies”: quench-based antibody probes that show antigen-dependent fluorescence.
Abe R, Ohashi H, Iijima I, Ihara M, Takagi H, Hohsaka T, Ueda H.
J Am Chem Soc. 2011 Nov 2;133(43):17386-94.

3) Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells.
Hiroshima M, Saeki Y, Okada-Hatakeyama M, Sako Y.
Proc Natl Acad Sci U S A. 2012 Aug 28;109(35):13984-9.

4) Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein.
Nagai H, Ebisu S, Abe R, Goto T, Takahashi N, Hosaka T, Kawada T.
Biosci Biotechnol Biochem. 2011;75(2):337-41.

5) Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence.
Abe R, Jeong HJ, Arakawa D, Dong J, Ohashi H, Kaigome R, Saiki F, Yamane K, Takagi H, Ueda H.
Sci Rep. 2014 Apr 11;4:4640.

6) Strategy for making a superior Quenchbody to proteins: effect of the fluorophore position.
Jeong HJ, Ueda H.
Sensors (Basel). 2014 Jul 23;14(7):13285-97.

7) In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.
Tada S, Zang Q, Wang W, Kawamoto M, Liu M, Iwashita M, Uzawa T, Kiga D, Yamamura M, Ito Y.
J Biosci Bioeng. 2015 Feb;119(2):137-9.

8) A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid.
Wang W, Uzawa T, Tochio N, Hamatsu J, Hirano Y, Tada S, Saneyoshi H, Kigawa T, Hayashi N, Ito Y, Taiji M, Aigaki T, Ito Y.
Chem Commun (Camb). 2014 Mar 18;50(22):2962-4.

9) Genetic PEGylation.
Tada S, Andou T, Suzuki T, Dohmae N, Kobatake E, Ito Y.
PLoS One. 2012;7(11):e49235.

10) In vitro selection of a photo-responsive peptide aptamer using ribosome display.
Liu M, Tada S, Ito M, Abe H, Ito Y.
Chem Commun (Camb). 2012 Dec 18;48(97):11871-3.

11) A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.
Ezure T, Nanatani K, Sato Y, Suzuki S, Aizawa K, Souma S, Ito M, Hohsaka T, von Heijine G, Utsumi T, Abe K, Ando E, Uozumi N.
PLoS One. 2014 Dec 8;9(12):e112874.

12) Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist.
Goto T, Nagai H, Egawa K, Kim YI, Kato S, Taimatsu A, Sakamoto T, Ebisu S, Hohsaka T, Miyagawa H, Murakami S, Takahashi N, Kawada T.
Biochem J. 2011 Aug 15;438(1):111-9.

13) Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein.
Nagai H, Ebisu S, Abe R, Goto T, Takahashi N, Hosaka T, Kawada T.
Biosci Biotechnol Biochem. 2011;75(2):337-41.

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