title: Expression, purification and characterization of heterotrimeric forms of sTRAIL using a polycistronic expression vector.
abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is capable of selectively inducing apoptosis of cancer cells, is a potential targeted drug for cancer therapy. The TRAIL protein induces apoptosis only in trimeric form. However, the recombinant soluble TRAIL (sTRAIL) trimer has low stability and a short half-life, which is a major obstacle for its advancement into clinical trials. Moreover, a percentage of engineered sTRAIL proteins are produced as dimers which may be toxic to normal human hepatocytes. In this study, we inserted three copies of the same subunit fragment of sTRAIL with a His tag into a polycistronic expression vector (pST39) to explore whether it would increase the proportion of trimers. We also constructed a heterozygous vector containing three subunit fragments of sTRAIL each with a different tag (His, HA, and Cmyc). Hybrid sTRAIL proteins (P-dTags) mainly as heterologous trimers were obtained by elution with a low concentration of imidazole based on different binding affinities of His with a nickel column. Functional analysis demonstrated that heterotrimeric forms of sTRAIL showed more stable activity compared to the P-3H at 4°C but not at 37°C without alteration in the native killing capacity. In addition, the heterologous trimers showed decreased toxicity to hepatocytes. These results suggest that the polycistronic expression system may be useful for expression of recombinant sTRAIL and improving its potential in cancer therapeutic applications.
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Baoming
authors.initials: B
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Yan
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Lizheng
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Zixuan
authors.initials: Z
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China. Electronic address: yubin@jlu.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Yu
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China. Electronic address: xianghui@jlu.edu.cn.
authors.forename: Xianghui
authors.initials: X
authors.lastname: Yu
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D002470:Cell Survival; D004926:Escherichia coli; D006801:Humans; D010957:Plasmids / Q000235:genetics; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand
keywords: Polycistronic expression system; TARIL; Tag; Trimer