Difficulties were encountered when trying to express human collagen
Barley (Hordeum vulgare).
Barley plants were transformed with eight different genetic constructs.
Two constructs contained a version of the rCIA1 gene for human
collagen I alpha-1. The remaining six constructs contained only part
of the rCIA1 gene that codes for a fragment of the larger
collagen protein. Three different promoters were tested: a maize
ubiquitin promoter (Ubi-1), which is constitutively active throughout
the plant; a seed (endosperm)-specific promoter from the rice glutelin
gene; and an alpha-amylase promoter from barley, which is active during
"The pharmaceutical and food industries use collagen and its derivative
gelatin (a denatured form of collagen) extensively in various
applications. . . . The traditional way of gelatin
production is by extraction from collagen-rich tissues of
animals. . . . Using recombinant technology, it
may be possible to design and express gelatin molecules with predetermined
characteristics to match the requirements in a variety of pharmaceutical
applications" (p. 657-8).
Goal of This Study:
Explore the "feasibility of using transgenic barley seeds as an expression
system for recombinant collagen-related protein products" (p. 658).
Several transgenic barley lines accumulated high levels of the human
collagen fragment in their seeds.
Microprojectile bombardment of barley cells was attempted with six
genetic constructs, but transgenic plants could be created only with
three of these for reasons that were unknown.
The full-length collagen protein was found at low but detectable levels
in the seeds of five transgenic (Ubi-1) plants regenerated from tissue
culture, but the next generation had fertility problems (low yield)
and produced seeds with no detectable human collagen.
The collagen fragment protein was purified from one of the transgenic
lines and analyzed for certain modifications. In humans, roughly half
the proline amino acids in collagen are modified to hydroxyproline, which
is required for the formation of collagen's unique triple helix structure.
The transgenic collagen fragment in barley had only 10% of its proline
residues hydroxylated. When the purified collagen fragment was analyzed,
three different versions of it were detected, possibly due to inconsistent
patterns of proline hydroxylation.
In addition to propagating the transgenic barley plants by seed,
a procedure known as doubled haploid breeding was used to create
plants with two copies of the collagen gene. However, doubled haploid
plants could not be regenerated for one of the more promising transgenic
lines, for reasons that were unknown.
Eskelin, K., A. Ritala, T. Suntio, S. Blumer et al. (2009). "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 Biotechnology
Journal vol. 7, pp. 657-72.
University of Helsinki, Finland; VTT Research Centre, Finland; FibroGen
Technology Development Agency and Ministry of Agriculture, Finland.
Not on the market as of 2009.
Copyright 2009 The Nature