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.
Plant Biotechnol J
; 7(7): 657-72, 2009 Sep.
Article
em En
| MEDLINE
| ID: mdl-19656332
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Peptídeos
/
Sementes
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Hordeum
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Proteínas Recombinantes
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Plantas Geneticamente Modificadas
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Colágeno Tipo I
Limite:
Humans
Idioma:
En
Revista:
Plant Biotechnol J
Assunto da revista:
BIOTECNOLOGIA
/
BOTANICA
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Finlândia