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

Katri Eskelin, Anneli Ritala, Taina Suntio, Susan Blumer, Heidi Holkeri, Eva H. Wahlström, Julio Baez, Kristiina Mäkinen (Corresponding Author), Anna-Maria Nuutila

Research output: Contribution to journalArticleScientificpeer-review

28 Citations (Scopus)

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 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.
Original languageEnglish
Pages (from-to)657-672
Number of pages6
JournalPlant Biotechnology Journal
Volume7
Issue number7
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Hordeum
Collagen Type I
collagen
Seeds
barley
glutelins
promoter regions
seeds
Glutens
Haploidy
doubled haploids
Amylases
ubiquitin
Ubiquitin
amylases
genetically modified organisms
recombinant DNA
Recombinant DNA
structural proteins
post-translational modification

Keywords

  • collagen
  • gelatin
  • transgenic barley
  • recombinant protein
  • seed-specific expression
  • plant molecular farming
  • endoplasmic reticulum targeting

Cite this

Eskelin, Katri ; Ritala, Anneli ; Suntio, Taina ; Blumer, Susan ; Holkeri, Heidi ; Wahlström, Eva H. ; Baez, Julio ; Mäkinen, Kristiina ; Nuutila, Anna-Maria. / 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. In: Plant Biotechnology Journal. 2009 ; Vol. 7, No. 7. pp. 657-672.
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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 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.",
keywords = "collagen, gelatin, transgenic barley, recombinant protein, seed-specific expression, plant molecular farming, endoplasmic reticulum targeting",
author = "Katri Eskelin and Anneli Ritala and Taina Suntio and Susan Blumer and Heidi Holkeri and Wahlstr{\"o}m, {Eva H.} and Julio Baez and Kristiina M{\"a}kinen and Anna-Maria Nuutila",
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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. / Eskelin, Katri; Ritala, Anneli; Suntio, Taina; Blumer, Susan; Holkeri, Heidi; Wahlström, Eva H.; Baez, Julio; Mäkinen, Kristiina (Corresponding Author); Nuutila, Anna-Maria.

In: Plant Biotechnology Journal, Vol. 7, No. 7, 2009, p. 657-672.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - 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

AU - Eskelin, Katri

AU - Ritala, Anneli

AU - Suntio, Taina

AU - Blumer, Susan

AU - Holkeri, Heidi

AU - Wahlström, Eva H.

AU - Baez, Julio

AU - Mäkinen, Kristiina

AU - Nuutila, Anna-Maria

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N2 - 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.

AB - 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.

KW - collagen

KW - gelatin

KW - transgenic barley

KW - recombinant protein

KW - seed-specific expression

KW - plant molecular farming

KW - endoplasmic reticulum targeting

U2 - 10.1111/j.1467-7652.2009.00432.x

DO - 10.1111/j.1467-7652.2009.00432.x

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