Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes

Takuma Ohtsuka; Satoshi Neki; Tamotsu Kanai; Kazunari Akiyoshi; Shin Ichiro M. Nomura; Takashi Ohtsuki

doi:10.1016/j.ab.2011.06.026

Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes

Takuma Ohtsuka, Satoshi Neki, Tamotsu Kanai, Kazunari Akiyoshi, Shin Ichiro M. Nomura, Takashi Ohtsuki

Research output: Contribution to journal › Article › peer-review

14 Citations (Scopus)

Abstract

The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.

Original language	English
Pages (from-to)	97-101
Number of pages	5
Journal	Analytical Biochemistry
Volume	418
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2011.06.026
Publication status	Published - 2011 Nov 1
Externally published	Yes

Keywords

Fluorescence correlation spectroscopy
Four-base codon
Liposome
Membrane protein
Protein labeling
Translation

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.ab.2011.06.026

Cite this

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title = "Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes",

abstract = "The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.",

keywords = "Fluorescence correlation spectroscopy, Four-base codon, Liposome, Membrane protein, Protein labeling, Translation",

author = "Takuma Ohtsuka and Satoshi Neki and Tamotsu Kanai and Kazunari Akiyoshi and Nomura, {Shin Ichiro M.} and Takashi Ohtsuki",

note = "Funding Information: This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Bio-manipulation” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (20034036 to T.O.). We thank A. Saito and H. Nakata (Olympus) for technical help. ",

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doi = "10.1016/j.ab.2011.06.026",

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AU - Ohtsuka, Takuma

AU - Neki, Satoshi

AU - Kanai, Tamotsu

AU - Akiyoshi, Kazunari

AU - Nomura, Shin Ichiro M.

AU - Ohtsuki, Takashi

N1 - Funding Information: This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Bio-manipulation” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (20034036 to T.O.). We thank A. Saito and H. Nakata (Olympus) for technical help.

PY - 2011/11/1

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N2 - The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.

AB - The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.

KW - Fluorescence correlation spectroscopy

KW - Four-base codon

KW - Liposome

KW - Membrane protein

KW - Protein labeling

KW - Translation

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JO - Analytical Biochemistry

JF - Analytical Biochemistry

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ER -

Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes

Abstract

Keywords

ASJC Scopus subject areas

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