Production of a recombinant industrial protein using barley cell cultures.
The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.
Ritala
A
A
VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 Espoo, VTT, Finland. anna-maria.nuutila@vtt.fi
Wahlström
E H
EH
Holkeri
H
H
Hafren
A
A
Mäkeläinen
K
K
Baez
J
J
Mäkinen
K
K
Nuutila
A M
AM
D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques; D024042:Collagen Type I / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001467:Hordeum / Q000166:cytology / Q000235:genetics*; D006801:Humans; D010434:Pepsin A / Q000737:chemistry; D010843:Pichia / Q000235:genetics; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D024042:Collagen Type I; D011994:Recombinant Proteins; D010434:Pepsin A