Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.
Previously, we established a method to produce recombinant proteins (r-proteins) in cocoons of germline transgenic silkworms, and showed that a step(s) in post-transcription processes was rate-limiting in obtaining a high yield of r-proteins. In this study, we examined whether the 5'-untranslated region (5'-UTR) of the polyhedrin gene (pol) of nucleopolyhedrovirus (NPV) has a translational enhancer activity in the r-protein expression by middle silk gland (MSG) cells of silkworm Bombyx mori (Bm). Sericin 1 gene (ser1) promoter-driven transformation vectors were constructed in which pol5'-UTRs of NPVs isolated from four different species, Bm, Spodoptera frugiperda, Ectropis oblique, and Malacosoma neustria, were each placed upstream of a reporter gene. Transient expression assays in MSGs showed that these pol5'-UTRs all enhanced the protein expression of reporter genes, and the pol5'-UTR of Bm NPV (pol5'-UTR/Bm) was the most effective among them. Thus, transgenic silkworms were generated, which bore the ser1 promoter-driven His-tagged secretory EGFP (sEGFP-His) gene under the control of pol5'-UTR/Bm. The synthesis of sEGFP-His proteins in MSGs of the transgenic worms was approximately 1.5-fold higher than that in those bearing null vectors. However, its mRNA expression levels were 67% of the control worms, indicating that the pol5'-UTR/Bm specifically enhanced the translational level. In conclusion, pol5'-UTR/Bm increased the yield of r-protein production in transgenic silkworms by enhancing the translational activity and this 5'-UTR could be useful for the mass production of r-proteins in germline transgenic silkworms.
Iizuka
Masashi
M
Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.
Tomita
Masahiro
M
Shimizu
Katsuhiko
K
Kikuchi
Yutaka
Y
Yoshizato
Katsutoshi
K
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism*; D012831:Bombyx / Q000235:genetics / Q000821:virology*; D004268:DNA-Binding Proteins / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D019476:Insect Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis*
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C085605:polyhedrin promoter-binding protein, insect