Team:Kyoto/CiC/Method

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Method

Method for Subgoal 1 --- Liposomes with HIV-1 TAT peptide

Subgoal A is construction of liposome that penetrate through human cell membranes. To acheive this, we designed liposomes with HIV-1 TAT peptide.

HIV-1 TAT is a Protein Transduction Domain(PTD)[2]. Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane[1].

It is suggested that HIV-1 TAT promotes endocytosis,though the precise mechanism remains unclear.

Then we fused HIV-1 TAT with potential anchor protein (LALAAAALALAAAALALAAAA)[3]. The LALAAAA side of this fusion protein is expected to be anchored in the lipid barrier of liposome. And the TAT side of the protein will be exposed to water so that TAT domain can touches the cell membrane. When exposed to cells, liposomes with this fusion protein will promote endocytosis and be taken into the cell. If such liposomes are fluorescent stained, they will show fluorescence in the cells.

We constructed plasmids encoding this fusion protein. When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes[4].

We also performed similar experiments with chemically synthesized TAT-LALAAAA protein.

Method for Subgoal 2 --- Liposomes with Mitochondrial Translocases

Method for Subgoal 3 --- Liposomes with HIV-1 TAT and Mitochondrial Translocases

reference

Subgoal 1

[1] Nobuhiro Yagi: Furukute atarashi miwakuno nanoryuusi Liposome(Classic yet new and attractive nanoparticle:liposome).yakuzaigaku, 68 (5),(2008).

[2] Hideki MATSUI, Kazuhito TOMIZAWA and Masayuki MATSUSHITA: Protein transduction by poly-arginine.. Jpn.121，(2003)

[3] Yoshiaki Yano, Tomokazu Takemoto, Satoe Kobayashi, Hiroyuki Yasui, Hiromu Sakurai,Wakana Ohashi, Miki Niwa, Shiroh Futaki, Yukio Sugiura, and Katsumi Matsuzaki: Topological Stability and Self-Association of a Completely Hydrophobic Model Transmembrane Helix in Lipid Bilayers.Biochemistry, 2002, 41(9)

[4] Shin-ichiro M. Nomura, Satoshi Kondoha, Wakiko Asayamaa, Akikazu Asada, Shigemichi. Nishikawa and Kazunari Akiyoshia: Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis.Journal of Biotechnology Volume 133, Issue 2, 20 January 2008.

Subgoal 2

Subgoal 3

@@ Line 31: / Line 31: @@
 To acheive this, we designed ''' liposomes with HIV-1 TAT peptide.'''
-HIV-1 TAT is a Protein Transduction Domain(PTD).[2]
+HIV-1 TAT is a Protein Transduction Domain(PTD)[2].
-Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane.[1]
+Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane[1].
-It is suggested that HIV-1 TAT promotes endocytosis,though the precise mechanism remains unclear.
+It is suggested that HIV-1 TAT promotes endocytosis,though the precise mechanism remains unclear.
-Then we fused HIV-1 TAT with potential anchor protein (LALAAAALALAAAALALAAAA).[3]
+Then we fused HIV-1 TAT with potential anchor protein (LALAAAALALAAAALALAAAA)[3].
 The LALAAAA side of this fusion protein is expected to be anchored in the lipid barrier of liposome.
 And the TAT side of the protein will be exposed to water so that TAT domain can touches the cell membrane.
@@ Line 42: / Line 43: @@
 We constructed plasmids encoding this fusion protein.
-When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes.[4]
+When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes[4].
 We also performed similar experiments with chemically synthesized TAT-LALAAAA protein.