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

Abstract

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).

Detection and expression analysis of recombinant proteins in plant‐derived complex mixtures using nanoUPLC‐MSE

“…The percentages of each detected protein are listed in Table 2. CTAG had an expression value of 0.1%, which is low compared with that of the other transgenic soybean seeds that express hGH [9] (2.9%), but it had a similar value compared with factor IX expression (0.2%) [10]. Other soybean proteins, such as b-conglycinin and glycinin, have been shown to exhibit the expected values that would be predicted for these storage proteins in soybean seeds [53].…”

“…Seeds are an advantageous production platform for recombinant proteins for several reasons: they can undergo long-term storage at ambient temperatures [5,6], they can provide an appropriate biochemical environment for protein stability through the creation of specialized storage compartments [6,7], they are not contaminated by human or animal pathogens [8], their desiccation characteristics prevent them from undergoing non-enzymatic hydrolysis or protease degradation [5,8], and they do not carry the harmful substances that are present in some plant leaves, which is important for downstream processing [3,8]. We recently produced several transgenic soybean plants that express important pharmaceutical molecules, such as proinsulin [6], human growth hormone (hGH) [9] and human coagulation factor IX (hFIX) [10], which demonstrate the versatility of this system. However, the production of these transgenic lines is extremely time consuming [11] and requires at least 150 days to obtain the first seeds and another three years to generate a homozygous line.…”

Advancements in Transgenic Soy: From Field to Bedside

“…While this treatment is effective at preventing and arresting hemorrhage, it is very costly and the protein is difficult to produce in large quantities. Using a biolistic transformation approach, hFIX expression was targeted to soybean seeds using the soy 7S promoter and coixin signal peptide [75]. Recombinant hFIX protein accumulated to 0.23% of seed TSP, and purified protein exhibited blood-clotting activity up to 1.…”

Coagulation Factor IX for Hemophilia B Therapy

“…Expression of inactive FIX in transgenic plants has been reported to date in transgenic tomatoes [61] and soybean seeds [62]. The expression level of FIX in tomatoes was quite low: 15.84 µg/kg of fresh fruits.…”