Abstract
The extracellular portion (amino acids 95–281 or 114–281) of the human tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) was genetically linked to the C terminus of the fluoresce-enhanced green fluorescent protein variant (EGFP) to generate two versions of EGFP–sTRAIL fusion proteins, designated EGFP–sTR95 and EGFP–sTR114, respectively. The two versions of EGFP–sTRAIL fusion proteins both induce extensive apoptosis in lymphoid as well as nonlymphoid tumor cell lines. In addition, the two versions of fusion proteins retain similar fluorescence spectra to those of EGFP and have shown the specific binding to TRAIL receptor-positive cells; thus, the stained cells could be analyzed with flow cytometry. Hence, the two versions of fusion proteins represent a readily obtainable source of biologically active sTRAIL that may prove useful in exploit fully the characteristics of both the soluble TRAIL and its receptor system.
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Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, Blackie C, Chang L, McMurtrey AE, Hebert A, DeForge L, Koumenis IL, Lewis D, Harris L, Bussiere J, Koeppen H, Shahrokh Z, Schwall RH (1999) Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104:155–162
Cao P, Zhang S, Zhang J, Wang M (2006) Construction and characterization of a bi-functional EGFP/sBAFF fusion protein. Biochimie 88:629–635
Chalfie M (1995) Green fluorescent protein. Photochem Photobiol 60:651–656
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805
Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L (1997) Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kB pathway. Immunity 7:821–830
Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene (Amst) 173:33–38
Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455
Degli-Esposti MA, Smolak PJ, Walczak H, Waugh J, Huang CP, DuBose RF, Goodwin RG, Smith CA (1997) Cloning and characterization of TRAIL-R3 a novel member of the emerging TRAIL receptor family. J Exp Med 186:1165–1170
Di Pietro R, Secchiero P, Rana R, Gibellini D, Visani G, Bemis K, Zamai L, Miscia S, Zauli G (2001). Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by selective up-regulation of TRAIL-R1. Blood 97:2596–2603
Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appelbaum ER, Eichman C, DiPrinzio R, Dodds RA, JamesIE, Rosenberg M, Lee JC, Young PR (1998) Osteoprotegerinis a receptor for the cytotoxic ligand TRAIL. J Biol Chem 273:14363–14367
Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91:12501–12504
Jo M, Kim TH, Seol DW, Esplen JE, Dorko K, Billiar TR, Strom SC (2000) Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nat Med 6:564–567
Kayagaki N, Yamaguchi N, Nakayama M, Eto H, Okumura K, Yagita H (1999a) Type I interferons (IFNs) regulate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on human T cells: a novel mechanism for the antitumor effects of type I IFNs. J Exp Med 189:1451–1460
Kayagaki N, Yamaguchi N, Nakayama M, Kawasaki A, Akiba H, Okumura K, Yagita H (1999b) Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity. J Immunol 162:2639–2647
Kayagaki N, Yamaguchi N, Nakayama M, Takeda K, Akiba H, Tsutsui H, Okamura H, Nakanishi K, Okumura K, Yagita H (1999c) Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells. J Immunol 163:1906–1913
Laemmli UK (1970) Cleavage of structural proteins during assembly of the heat of bacteriophage T4. Nature 277:680–685
Marshall J, Molloy R, Moss GW, Howe JR, Hughes TE (1995) The jellyfish green fluorescent protein, a new tool for studying ion channel expression and function. Neuron 14:211–215
Martinez-Lorenzo MJ, Alava MA, Gamen S, Kim KJ, Chuntharapai A, Pinerio A, Naval J, Anel A (1998) Involvement of APO2/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells. Eur J Immunol 28:2174–2725
Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM (1997a) An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277:815–818
Pan G, O’Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM (1997b) The receptor for the cytotoxic ligand TRAIL. Science 276:111–113
Pan G, Ni J, Yu G, Wei YF, Dixit VM (1998) TRUNDD, a new member of the TRAIL receptor family that antagonizes TRAIL. FEBS Lett 424:41–45
Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271:12687–12690
Rosorius O, Heger P, Stelz G, Hirschmann N, Hauber J, Stauber RH (1999) Direct observation of nucleo-cytoplasmic transport by microinjection of GFP-tagged proteins in living cells. Biotechniques 27:350–355
Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schooley KA, Smith CA, Goodwin RG, Rauch CT (1997) TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J 16:5386–5397
Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH (1999a) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5:157–163
Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH (1999b) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5:157–163
Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, Goodwin RG (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:673–682
Wu GS, Burns TF, McDonald ER, Jiang W, Meng R, Krantz ID, Kao G, Gan DD, Zhou JY, Muschel R, Hamilton SR, Spinner NB, Markowitz S, Wu G, El-Deiry WS (1997) KILLER /DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat Genet 17:141–143
Yang TT, Cheng L, Kain SR (1996) Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 24:4592–4593
Zamai L, Ahmad M, Bennett IM, Azzoni L, Alnemri ES, Perussia B (1998) Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J Exp Med 188:2375–2380
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This work was supported by grants from the Major State Basic Research Development Program of China (no. 2002CB513000) and National Nature Science Foundation of China (no. 30270527 and 30370556).
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Shen, J., Wu, Y., Shi, L. et al. Construction and characterization of two versions of bifunctional EGFP–sTRAIL fusion proteins. Appl Microbiol Biotechnol 76, 141–149 (2007). https://doi.org/10.1007/s00253-007-1001-1
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DOI: https://doi.org/10.1007/s00253-007-1001-1