Construction of tumor-specific toxins using ubiquitin fusion technique Sergey O. Tcherniuk, Jadwiga Chroboczek, Maxim Y. Balakirev  Molecular Therapy  Volume 11, Issue 2, Pages 196-204 (February 2005) DOI: 10.1016/j.ymthe.2004.10.009 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Construction of Ub-fusion-toxin chimeras. (A) Schematic representation of the pET11dUb-SAP expression constructs coding for UFT1–UFT3. The appropriate amino acid linker sequences with protease recognition sites are shown. The restriction enzymes used for initial saporin cloning into pET11d [19] are also indicated. (B) Western blot analysis of the cleavage of UFTs by ubiquitin-specific protease USP8 and PSA performed with polyclonal anti-saporin serum at 1:2500. The control lanes show the positions of purified recombinant proteins. Molecular Therapy 2005 11, 196-204DOI: (10.1016/j.ymthe.2004.10.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Stability of the saporin constructs in reticulocyte lysate. (A) Time course of in vitro ubiquitination and degradation of wild-type saporin (SO6; lanes 1–5), UFT1 (lanes 6–10), UFT2 (lanes 11–15), and UFT3 (lanes 16–20) analyzed by Western blotting with anti-saporin serum. In control experiments (lanes 1, 6, 11, 16) reticulocyte lysate was replaced by 40 mM Tris, pH 7.5. (B) Effects of Ubal and MG132 on ubiquitination and degradation of wild-type SO6 (lanes 1–4), UFT1 (lanes 5–8), UFT2 (lanes 9–12), and UFT3 (lanes 13–16) analyzed by Western blotting with anti-saporin antiserum. The reaction was stopped at 30 min. The reaction conditions were the same as in (A), with 5 μM Ubal or 50 μM MG132 present where indicated. Molecular Therapy 2005 11, 196-204DOI: (10.1016/j.ymthe.2004.10.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Inhibition of in vitro protein synthesis by saporin constructs. (A) Activity of luciferase expressed in vitro in reticulocyte lysate containing wild-type saporin (SO6 WT), UFT1, UFT2, or UFT3. The appropriate IC50 are shown at the bottom (mean values and ± SD, n = 3). (B–D) The same as in (A), but the lysate was preincubated with 50 μM MG132, 5 μM Ubal, or 10 μg/ml human PSA, respectively, for 15 min before addition of luciferase mRNA. Molecular Therapy 2005 11, 196-204DOI: (10.1016/j.ymthe.2004.10.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Cytotoxic activity of saporin constructs. (A) Inhibition of luciferase synthesis in transiently transfected HeLa, PC-3, and LNCaP cells by the indicated concentrations of UFTs. The activity is shown as RLU (mean values and ± SD, n = 3). Control column represents the activity of luciferase expressed in cells without toxin addition. The effect of wild-type saporin is not shown, because at the indicated concentrations it induced complete inhibition of luciferase synthesis. (B) Cytotoxicity of saporin constructs measured by the nonradioactive cell proliferation assay. HeLa, PC-3, and LNCaP cells were incubated in the presence of toxins for 48 h, whereupon MTS tetrazolium salt was added. ID50 refers to the concentration of toxin required to reduce the light-absorbance capacity of exposed cell cultures by 50%. Molecular Therapy 2005 11, 196-204DOI: (10.1016/j.ymthe.2004.10.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions