Production of a recombinant full-length collagen type I alpha-1 and of a 45-kDa collagen type I alpha-1 fragment in barley seeds.
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 alpha1 (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 alpha-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 alpha-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.
Eskelin
Katri
K
Department of Applied Chemistry and Microbiology and Department of Applied Biology, University of Helsinki, Helsinki, Finland.
Ritala
Anneli
A
Suntio
Taina
T
Blumer
Susan
S
Holkeri
Heidi
H
Wahlström
Eva H
EH
Baez
Julio
J
Mäkinen
Kristiina
K
Maria
Nuutila Anna
NA
D015153:Blotting, Western; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D000090602:Collagen Type I, alpha 1 Chain; D004797:Enzyme-Linked Immunosorbent Assay; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012639:Seeds / Q000235:genetics / Q000378:metabolism*
C000716127:COL1A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D010446:Peptide Fragments; D011994:Recombinant Proteins