Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds

• Published in: Transgenic research Vol. 20; no. 4; pp. 841 - 855
• Main Authors: Cunha, Nicolau B., Murad, André M., Ramos, Gustavo L., Maranhão, Andréia Q., Brígido, Marcelo M., Araújo, Ana Cláudia G., Lacorte, Cristiano, Aragão, Francisco J. L., Covas, Dimas T., Fontes, Aparecida M., Souza, Gustavo H. M. F., Vianna, Giovanni R., Rech, Elíbio L.
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.08.2011; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Animal Genetics and Genomics; Antigens, Plant - genetics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biotechnology; Blood Coagulation - drug effects; Coagulation factor IX; Embryos; Enzyme-linked immunosorbent assay; Factor IX - genetics; Factor IX - metabolism; Factor IX - pharmacology; Fundamental and applied biological sciences. Psychology; Gene transfer; Genetic Engineering; Genetic technics; Globulins - genetics; Glycine max; Glycine max - genetics; Glycine max - metabolism; Greenhouses; Humans; Immunocytochemistry; Life Sciences; Mass spectroscopy; Methods. Procedures. Technologies; Molecular Medicine; Molecular Sequence Data; Original Paper; Plant Genetics and Genomics; Plant Proteins - genetics; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Plasmids; Promoter Regions, Genetic; Promoters; Protein Sorting Signals - genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Regulatory sequences; Seed Storage Proteins - genetics; Seeds; Seeds - genetics; Seeds - metabolism; Signal peptides; Soybean Proteins - genetics; Soybeans; Temperature effects; Transcription; Transgenic animals and transgenic plants; Transgenics; Vacuoles; Western blotting; Transgenic soybean seeds; Human coagulation factor IX; Clotting assay; Biolistic-mediated transformation; Human; Microprojectile bombardment; Factor IX; Genetic transformation; Coagulation; Transgenic plant; Coagulation factor; Accumulation
• ISSN: 0962-8819; 1573-9368; 1573-9368
• DOI: 10.1007/s11248-010-9461-y

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [ Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α′ subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg −1 seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).