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Deletion of the GPI pre-anchor sequence in Mtf as a mechanism for generating the soluble form of the protein Yang, Joseph

Abstract

Melanotransferrin, also known as the human melanoma tumour associated antigen, p97, was one of the first cell surface markers associated with human skin cancer. p97 exists in two forms - the soluble form of the protein, and one that is attached to the cell membrane by a glycosylphosphatidyl-inositol (GPI) anchor. The p97 protein belongs to an important group of iron binding proteins that includes human transferrin (Tf), human lactoferrin and ovotransferrin from avian egg whites. Although there is high homology to these proteins, p97's biological function has yet to be determined. p97 may be involved in metal transport as it has been shown to bind and internalize iron, providing an alternative uptake mechanism of iron into the cell. p97 has also been shown to be associated with Alzheimer's Disease (AD), with elevated levels of p97 in the serum and those associated with amyloid plaques in patients afflicted with the disease. p97 may play a role in the pathology of this disease or the elevated levels may indicate a response to the pathophysiology of the disease. In addition to p97 as a marker for AD, it has been suggested that p97 may have the unique ability of traversing the blood brain barrier and provide a potential avenue for therapy by drug delivery into the brain. The exact mechanisms mediating this crossing, whether it is the GPI form of p97, or the soluble form binding to a putative receptor have not been determined. The need for further investigation in elucidating the function of p97 is clear. These experiments require substantial amounts of pure recombinant p97, consequently, an efficient method of generating the p97 molecule is required. The generation of p97 protein up to now, has involved labour intensive and costly procedures of purifying low levels of p97 present in the medium of cells expressing the full-length form (p97aTRVb c.3). The objective of this thesis has focused on generating the melanotransferrin (p97) protein in its soluble form for ease of purification and efficient generation of large amounts of p97. The approach involved sequential deletions of the p97 GPI pre-anchor sequence (PAS) up to the putative site of cleavage/attachment, releasing p97 from attachment to the GPI-anchor and rendering it soluble. Transfection of the p97 deletion constructs into both the CHO and BHK TK" cells were performed with the aim of optimizing the production of p97 by utilizing the cell characteristics unique to each cell line. Altering the GPI PAS resulted in the generation of the recombinant soluble form that was secreted at significantly higher rates than from the full-length expressing cell lines. Increases were from 22 x 10⁻⁹μg/cell/hr to 241 x 10⁻⁹mg/cell/hr for expression in the CHO cell system, and from 220 x 10⁻⁹μg/cell/hr to 4970 x 10⁻⁹μg/cell/hr for the BHK system.. Futhermore, there appeared to be differences in the secretion rates between the different deletions suggesting the need for closer examination of the C-terminus in achieving maximum production of the altered proteins.

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