Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast.
Different expression systems such as bacteria and mammalian cells have been used to produce pharmaceutical proteins. In recent years, the use of plants as bioreactors offers efficient and economical systems in recombinant protein production. Furthermore, because of the large number of plastid copies in plants, chloroplast engineering functions as an effective method to increase recombinant protein expression. Because the commercially available insulin for treatment does not contain C-peptide, which is of great importance for type 1 diabetic patients, the current study introduces the human proinsulin gene fused with protein A into the tobacco chloroplast genome using the biolistic method. To achieve homoplasmy, three rounds of selection and regeneration of transforming cells were performed on the medium that contained spectinomycin antibiotic and hormones. The PCR analysis indicated the presence of the proinsulin gene in transplastomic plants. The reverse-transcription PCR analysis confirmed the expression of the proinsulin-protein A fusion at the transcription level. Immunoblot assays of leaf-derived protein extracts confirmed that the target gene expression is up to 0.2% of the total soluble protein. Our study showed that protein A fusion is not as efficient as other reported fusions. The transplastomic plants were also confirmed for homoplasmy using Southern blot analysis.
Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
Melina
M
Yarbakht
Mokhtar
M
Jalali-Javaran
Maryam
M
Nikkhah
Mehdi
M
Mohebodini
D002736:Chloroplasts / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D018745:Genome, Plant / Q000235:genetics; D006801:Humans; D030821:Plants, Genetically Modified; D011384:Proinsulin / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000235:genetics*; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D011384:Proinsulin
biolistic method; chloroplast engineering; homologous recombination; transplastomic tobacco