Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein.
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.
Zhang
Jufeng
J
Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.
Wang
Zhanli
Z
Wei
Fang
F
Qiu
Wei
W
Zhang
Liangren
L
Huang
Qian
Q
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
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D043525:Cytosine Deaminase; C468827:codA protein, E coli