Recombinant DNA technology can be used to design and express collagen and gelatin‐related proteins with predetermined composition and structure. Barley seed was chosen as a production host for a recombinant full‐length collagen type I α1 (rCIa1) and a related 45‐kDa rCIa1 fragment. The transgenic barley seeds were shown to accumulate both the rCIa1 and the 45‐kDa rCIa1 fragment. Even when the amount of the rCIa1 was just above the detection threshold, this work using rCIa1 as a model demonstrated for the first time that barley seed can be used as a production system for collagen‐related structural proteins. The 45‐kDa rCI1a fragment expression, targeted to the endoplasmic reticulum, was controlled by three different promoters (a constitutive maize ubiquitin, seed endosperm‐specific rice glutelin and germination‐specific barley α‐amylase fusion) to compare their effects on rCIa1 accumulation. Highest accumulation of the 45‐kDa rCIa1 was obtained with the glutelin promoter (140 mg/kg seed), whereas the lowest accumulation was obtained with the α‐amylase promoter. To induce homozygosity for stable 45‐kDa rCIa1 production in the transgenic lines, doubled haploid (DH) progeny was generated through microspore culture. The 45‐kDa rCIa1 expression levels achieved from the best DH lines were 13 mg/kg dry seeds under the ubiquitin promoter and 45 mg/kg dry seeds under the glutelin promoter. Mass spectroscopy and amino acid composition analysis of the purified 45‐kDa rCIa1 fragment revealed that a small percent of prolines were hydroxylated with no additional detectable post‐translational modifications.
- transgenic barley
- recombinant protein
- seed-specific expression
- plant molecular farming
- endoplasmic reticulum targeting
Eskelin, K., Ritala, A., Suntio, T., Blumer, S., Holkeri, H., Wahlström, E. H., Baez, J., Mäkinen, K., & Nuutila, A-M. (2009). Production of a recombinant full‐length collagen type I α‐1 and of a 45‐kDa collagen type I α‐1 fragment in barley seeds. Plant Biotechnology Journal, 7(7), 657-672. https://doi.org/10.1111/j.1467-7652.2009.00432.x