Thesis
Development of transgenic barley expressing human type I collagen
Washington State University
Master of Science (MS), Washington State University
2004
Handle:
https://hdl.handle.net/2376/278
Abstract
Collagen I is the main protein found in the extracellular matrix. It consists of a heterotrimeric triple helix that aggregates to form a fibrillar structure. Eight specific enzymes are needed for correct folding and secretion of the protein to the extracellular space. The need to find a non-animal source for the production of collagen has led to the development of expression systems that are able to produce recombinant collagen. The main objective of this dissertation was to produce transgenic barley grains expressing the genes needed for production of hydroxylated procollagen in the endosperm. To prepare for reaching this objective, it was necessary to codon optimize the gene and express it in an eucaryotic system like Pichia pastoris for verification that the correct protein product was synthesized. Then Agrobacterium tumefaciens mediated transformation of immature zygotic embryos of barley could be initiated with the appropriate vectors. Accordingly, vectors were constructed for expression in Pichia pastoris and barley. Homotrimeric procollagen I and prolyl 4-hydroxylase were successfully expressed in Pichia pastoris, as shown by SDS-PAGE, Western blot and ELISA. Barley was transformed by Agrobacterium tumefaciens with vectors carrying the genes for collagen and both subunits of prolyl 4-hydroxylase. Green plants were selected with the aid of bialaphos resistance and PCR-tested with specific primers for the internal part of ([alpha])1 collagen I chain.
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Details
- Title
- Development of transgenic barley expressing human type I collagen
- Creators
- Claudia E. Osorio
- Contributors
- Diter von Wettstein (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Crop and Soil Sciences, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Identifiers
- 99900525007201842
- Language
- English
- Resource Type
- Thesis