Semisynthesis of a protein with cholesterol at the C-terminal, targeted to the cell membrane of live cells.
Various proteins are modified post-translationally to localize them at the cell membrane. Among them, hedgehog-family proteins are modified by cholesterol at the C-terminal. In this study, green fluorescent protein (GFP) modified with cholesterol (GFP-Chol) at the C-terminal was prepared semisynthetically and investigated. This semi-synthesis was performed using the following native chemical ligation: GFP-Cα-thioester was prepared using the intein-mediated thioester exchange reaction and was ligated to Cys-NH-diethylene glycol-NHCO-cholesterol in the presence of a detergent. After removal of the detergent, the GFP-Chol was applied to mouse live cells. Confocal laser fluorescent microscopy confirmed localization of GFP-Chol at the cell membrane. The findings suggest that modifying proteins with cholesterol at the C-terminal is useful for targeting the proteins to the cell membrane of live cells.
Teruya
Kenta
K
Department of Neurochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-Machi, Aoba-Ku, Sendai, Miyagi, 980-8575, Japan.
Nishizawa
Keiko
K
Doh-ura
Katsumi
K
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002477:Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D002784:Cholesterol / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002784:Cholesterol
J Clin Invest. 2002 Sep;110(5):591-6
12208857
Eur J Biochem. 2004 Feb;271(4):663-77
14764082
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6705-10
9618476
J Biol Chem. 2004 May 28;279(22):23790-6
15026415
J Biol Chem. 1999 Apr 23;274(17):12049-54
10207028
Angew Chem Int Ed Engl. 2005 Sep 5;44(35):5713-7
16059958
Chem Biol. 2007 Sep;14(9):994-1006
17884632
Biotechniques. 1999 Jul;27(1):110-4, 116, 118-20
10407673
Science. 1994 Nov 4;266(5186):776-9
7973629
J Biol Chem. 1997 Mar 7;272(10):6324-31
9045652
J Biol Chem. 1995 Dec 29;270(52):31391-6
8537413
J Am Chem Soc. 2007 Sep 19;129(37):11543-50
17715922
J Biol Chem. 2003 Nov 14;278(46):45563-9
12947110
J Biol Chem. 1996 Sep 6;271(36):22159-68
8703028
Nat Biotechnol. 2003 Aug;21(8):936-40
12858181
J Am Chem Soc. 2004 Dec 22;126(50):16368-78
15600338
Chembiochem. 2003 Apr 4;4(4):265-71
12672105
Annu Rev Biochem. 2000;69:923-60
10966479
Cell. 1987 Oct 23;51(2):229-40
2444340
Cell. 1992 Feb 7;68(3):533-44
1531449