The M4 insulator, the TM2 matrix attachment region, and the double copy of the heavy chain gene contribute to the enhanced accumulation of the PHB-01 antibody in tobacco plants

• Published in: Transgenic research Vol. 29; no. 2; pp. 171 - 186
• Main Authors: Ruiz, Yoslaine, Ramos, Pedro Luis, Soto, Jeny, Rodríguez, Meilyn, Carlos, Natacha, Reyes, Aneisi, Callard, Danay, Sánchez, Yadira, Pujol, Merardo, Fuentes, Alejandro
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-04-2020; Springer Nature B.V
• Subjects: Animal Genetics and Genomics; Antibodies - genetics; Antibodies - metabolism; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Bioreactors; Chromatin; Deoxyribonucleic acid; DNA; Gene Expression Regulation, Plant; Genetic Engineering; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - metabolism; Immunoglobulin Light Chains - genetics; Immunoglobulin Light Chains - metabolism; Insulator Elements; Life Sciences; Matrix attachment regions; Matrix Attachment Regions - genetics; Molecular Medicine; Monoclonal antibodies; Nicotiana - genetics; Nicotiana - growth & development; Original Paper; Plant Genetics and Genomics; Plants, Genetically Modified - genetics; Plants, Genetically Modified - growth & development; Prohibitins; Promoter Regions, Genetic; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Tobacco; Transcription; Transgenes; Transgenes - genetics; Transgenic plants; Transgenics; Plant expression; Tobacco; Insulator; Antibody; Matrix attachment region
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-019-00187-6

The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human pathogens, enhance the use of plants to generate complex therapeutic products such as monoclonal antibodies. However, stably transformed plants expressing antibodies normally have a poor accumulation of these proteins that probably arise from the negative positional effects of their flanking chromatin. The induction of boundaries between the transgenes and the surrounding DNA using matrix attachment regions (MAR) and insulator elements may minimize these effects. With the PHB-01 antibody as a model, we demonstrated that the insertion of DNA elements, the TM2 (MAR) and M4 insulator, flanking the transcriptional cassettes that encode the light and heavy chains of the PHB-01 antibody, increased the protein accumulation that remained stable in the first plant progeny. The M4 insulator had a stronger effect than the TM2, with over a twofold increase compared to the standard construction. This effect was probably associated with an enhancer-promoter interference. Moreover, transgenic plants harboring two transcriptional units encoding for the PHB-01 heavy chain combined with both TM2 and M4 elements enhanced the accumulation of the antibody. In summary, the M4 combined with a double transcriptional unit of the heavy chain may be a suitable strategy for potentiating PHB-01 production in tobacco plants.