title: Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein.
abstract: Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.
authors.affiliation: Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.
authors.forename: Jufeng
authors.initials: J
authors.lastname: Zhang
authors.forename: Zhanli
authors.initials: Z
authors.lastname: Wang
authors.forename: Fang
authors.initials: F
authors.lastname: Wei
authors.forename: Wei
authors.initials: W
authors.lastname: Qiu
authors.forename: Liangren
authors.initials: L
authors.lastname: Zhang
authors.forename: Qian
authors.initials: Q
authors.lastname: Huang
mesh_terms: D045744:Cell Line, Tumor; D003198:Computer Simulation; D043525:Cytosine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D043525:Cytosine Deaminase; C468827:codA protein, E coli