Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light.
Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that heterologous protein accumulation in chloroplasts could be hindered by post-transcriptional mechanisms not yet characterized. Here, we describe the development and characterization of transplastomic tobacco plants transformed with four different transformation vectors for the expression of human epidermal growth factor (hEGF). We showed that, although the corresponding transcript was present in all of the analyzed plants, hEGF could only be detected when fused to the first 186 amino acids of bacterial beta-glucuronidase (GUS). In addition, we observed that the expression levels of recombinant protein increased when plants were placed in the dark or when leaves were incubated in the presence of electron transport inhibitors, such as methyl viologen (MV) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that the mechanism responsible for hEGF instability in chloroplasts is regulated by light.
Wirth
Sonia
S
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, INGEBI-CONICET, and FCEN-UBA, Vuelta de Obligado 2490, Buenos Aires, Argentina.
Segretin
Maria Eugenia
ME
Mentaberry
Alejandro
A
Bravo-Almonacid
Fernando
F
D015152:Blotting, Northern / Q000379:methods; D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008027:Light; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D005966:Glucuronidase