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

Abstract

SummaryRecombinant 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 a1 (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 a-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 a-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.

Barley

“…Western blot analysis of leaf tissue from T 0 plants documented the expression of the recombinant human lactoferrin (Kamenarova et al 2007 ), whereas Tanasienko et al ( 2011 ) provided proof for the presence of the gene hLF fragment in leaves of T 0 plants by PCR. Eskelin et al ( 2009 ) and Erlendsson et al ( 2010 ) presented further convincing evidences that transgenic barley seeds can be utilised as a bioreactor. The successful expression of both the recombinant full-length and the 45-kDa fragment of human collagen-type I α-1 chain ( rCla1 ) in barley seeds was obtained screening three promoters.…”

“…The proteins were further targeted to the endoplasmic reticulum to enhance the expression levels of recombinant proteins as previously shown (Horvath et al 2000 ). The glutelin promoter was superior in yielding 45 mg recombinant protein per kg dry seeds in the best lines compared to 15 mg/kg caused by the ubiquitin promoter (Eskelin et al 2009 ). Moreover, the Orfeus TM expression system developed by ORF Genetics (Reykjavik, Iceland) was used to produce recombinant human Flt3 ligand in barley grains.…”

TGP – the Database on Promoters for Plant Transgenesis

“…Этот промотор может быть с успехом использован в сельском хозяйстве при создании форм с мужской стерильностью. В некоторых случаях показано, что такая тканеспецифическая экспрессия является более выгодной, чем конститутивная, которая обеспечивается промоторами 35S вируса мозаики цветной капусты или убиквитина кукурузы [29][30][31]. Развитие новых методов для повышения устойчивости растений к различным стрессам также требует использование промоторов со специфическим паттерном экспрессии.…”

Bioengineered collagens

“…Notable progress has been made on commercial, recombinant production of human collagens. 7,[10][11][12][13][14][15][16][17] However, the rapidly emerging technologies that have characterized and produced recombinant non-animal (bacterial) collagens in E. coli [23][24][25]27,28 have introduced an exciting new collagen option that can be readily customized for specific biomedical applications. 28,33 These customized collagens will pave the way for more diverse applications in medical devices, implantable materials and potentially as scaffolds for stem cells, to name but a few examples of uses for these recombinant bacterial collagens.…”

“…Initial reports produced unhydroxylated or poorly hydroxylated collagen from leaves 15 and from seeds. 16 Additional introduction of the mammalian P4H is necessary for a quality collagen product, even though plants contain a form of P4H. Most recently, heterotrimeric collagen has been produced with P4H also present, giving excellent protein yields, which can be commercialized.…”