Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.
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).
Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica (PqEB), Av. W5 Norte, Brasília, DF 70770-917, Brazil.
Nicolau B
NB
Cunha
André M
AM
Murad
Gustavo L
GL
Ramos
Andréia Q
AQ
Maranhão
Marcelo M
MM
Brígido
Ana Cláudia G
AC
Araújo
Cristiano
C
Lacorte
Francisco J L
FJ
Aragão
Dimas T
DT
Covas
Aparecida M
AM
Fontes
Gustavo H M F
GH
Souza
Giovanni R
GR
Vianna
Elíbio L
EL
Rech
D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000235:genetics; D001777:Blood Coagulation / Q000187:drug effects; D005164:Factor IX / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005916:Globulins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D055314:Seed Storage Proteins / Q000235:genetics; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D030262:Soybean Proteins / Q000235:genetics; D013025:Glycine max / Q000235:genetics / Q000378:metabolism*
D052179:Antigens, Plant; D005916:Globulins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; C072398:alpha-coixin protein, Coix lacryma-jobi; D005164:Factor IX