School of Botany - Theses
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ItemThe production of foreign peptides and proteins in plant-cell culture(University of Melbourne, 1994)There is growing demand from medicine, industry and agriculture for large quantities of purified peptides and proteins. This thesis describes the use of cultured plant cells for the production of peptides and proteins from gene constructs introduced by transformation. The culture is a Nicotiana plumbaginifolia cell line (NPT5120) grown as callus and as suspension-cell cultures. Initially we were interested in the expression of analogues (denoted AVPA, AVP-G) of the mammalian peptide hormone arginine vasopressin (AVP). Peptide was secreted by the transformed cells but was unstable in the culture and did not accumulate to high levels (-77 pg/20 ml culture). This problem was partially solved by the addition of the protease inhibitor, bacitracin, however, bacitracin did not completely prevent the loss of the peptide and it retarded plant cell growth. In a further attempt to stabilize the peptide, it was incorporated within the S2-RNase from N. alata because the S2-RNase is stable in the extracellular environment of pistils, and in the presence of protein extracts from the plant-cell cultures. The S-RNases of solanaceous species have a hypervariable region which was replaced with the peptide analogue in these studies. The transformed plant cells produced a transcript (-940 bases) encoding the hybrid RNase at a level 30-fold lower than the level of transcript encoding the S2-RNase in styles of N. alata. Although hybrid transcript was detected readily, the hybrid-RNase was not detected, indicating that the modification at the hypervariable region was not tolerated. A similar result was obtained when the cells were transformed with a construct encoding the mature protein from the major house dust mite allergen (Der p 1) from Dermatophagoides pteronyssinus. The allergen did not accumulate in the plant culture even though transcript encoding Der p 1 was detected. It is likely that translation and/or post-translational processing was inefficient or absent. In contrast, when the cell line was used for the expression of an unmodified plant protein, the proteinase inhibitor (PI) from N. alata, PI protein accumulated in the transformed cells to detectable levels, -0.01% of the total protein. Thus the N. plumbaginifolia plant-cell culture has potential for the expression of foreign peptides and proteins but a better understanding of translation, co- and post-translational processes together with product stability is required before this system can be used to produce large quantities of foreign peptides and proteins.