Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

Li Tian,Samuel Sm Sun

doi:10.1186/1472-6750-11-91

Abstract

BackgroundHuman 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.ResultsIn 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.ConclusionsIn 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.

Highlights

Human granulocyte colony-stimulating factor is an important human cytokine which has been widely used in oncology and infection protection
For Construct SH, hGCSF was synthesized with the same molecular weight (MW) of 18.6 kD as commercially available Human granulocyte colony-stimulating factor (hG-colony stimulating factor (CSF)) produced in E.coli (Figure 2B)
The results of immunoblot analysis (Figure 2B) showed that the synthesized hG-CSF was of 18.6 kD, indicating both ubiquitin and signal peptide were absent in the final protein product

Summary

Introduction

Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. Hong et al [16] expressed recombinant hG-CSF in rice cell suspension culture with a maximum yield of 2.5 mg/L after 13 days incubation, but the expression in cell culture was not stable and dropped quickly. Besides this rice cell culture example, to our knowledge, there was only one report [17] on the expression of hG-CSF in plant, in which hGCSF was expressed in the amount of 500 mg/kg fresh tobacco leaves through a transient expression system. In previous studies in an attempt to express hG-CSF in plants, we experienced only low levels of its expression (lower than 0.05% total soluble protein) in transgenic Arabidopsis and tobacco [18]

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Results

Discussion

Conclusion