A novel platform for biologically active recombinant human interleukin-13 production.
Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.
Wang
David J
DJ
A.B. Lucas Secondary School, 656 Tennent Avenue, London, ON, Canada.
Brandsma
Martin
M
Yin
Ziqin
Z
Wang
Aiming
A
Jevnikar
Anthony M
AM
Ma
Shengwu
S
D015152:Blotting, Northern; D015153:Blotting, Western; D001826:Body Fluids; D045744:Cell Line, Tumor; D004269:DNA, Bacterial / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D018793:Interleukin-13 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D004269:DNA, Bacterial; D018793:Interleukin-13; D012333:RNA, Messenger; D011994:Recombinant Proteins; C036483:T-DNA; D014357:Trypsin