title: Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco.
abstract: BACKGROUND
  Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application.
  
  
  RESULTS
  In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells.
  
  
  CONCLUSIONS
  In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants.
authors: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors: Li
authors: L
authors: Tian
authors: Samuel Sm
authors: SS
authors: Sun
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D016179:Granulocyte Colony-Stimulating Factor