Processing and antigenicity of tag-linked glycoproteins expressed in mammalian cells
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The work presented within this thesis expands upon the theme within this laboratory, of utilising epitope-labelled recombinant proteins for the construction of multivalent subunit vaccines. Mammalian-cell expression- vectors were constructed which encoded a 14 amino acid epitope-tag, termed Pk-tag. The genes encoding the haemagglutinin (PIN) and fusion (F) glycoproteins (model type II and type I proteins respectively) from the paramyxovirus simian virus 5 (SV5), were inserted into the above vectors such that the sequence encoding the Pk-tag was present at the amino (N) or carboxy (C) terminus of SV5 HN, and the C-terminus of SV5 F. The genes were expressed in mammalian cells by utilising the vaccinia virus/T7 transient-expression system. Encouraging results were obtained which demonstrated that the addition of the Pk-tag to the N or C termini of SV5 HN, or to the C-terminus of SV5 F, did not prevent the production of; full length, N-linked glycosylated, oligomeric, natively folded and cell-surface localised Pk-tagged protein. An attempt was made to produce secretable forms of Pk-tagged SV5 HN and F. For this purpose, a vector was constructed which encoded a truncated version of the SV5 F protein in which the C-terminal transmembrane anchor & cytoplasmic tail were deleted, but which still possessed the sequence encoding the C-terminal Pk-tag at the C-terminus of the ectodomain. Expression of this gene in mammalian cells resulted in the production of a protein which had undergone N-linked glycosylation and partial oligomerisation. No secretion of the truncated Pk-tagged protein into the external milieu was detected. Furthermore, production of potentially secretable forms of N & C- terminally Pk-tagged SV5 HN was achieved by the construction of plasmid vectors in which the non-cleavable native HN signal sequence was replaced by a putative cleavable signal sequence from the Epstein Barr virus gp220/360 glycoprotein. Expression of the modified Pk-tagged HN genes in mammalian cells produced proteins of a lower mwt. than expected and which, apart from a small proportion of the N-terminally Pk-tagged molecules, did not possess N-linked oligosaccharides and were not recognised by conformationally sensitive mAbs. No secretion of the modified Pk-tagged HN into the external milieu was detected. Following the first initial characterisation of the Pk-tagged HN & F, in which very encouraging results were obtained, an attempt was made to isolate cell-lines which constitutively or inducibly expressed Pk-tagged HN. Production of Pk-tagged HN could not be detected from constructs in which expression was driven from constitutive promoters. However, production of N-terminally Pk-tagged HN (but not C-terminally tagged HN) was detected when expression was driven by the tTa inducible expression system. As a further development to the tTa system, a 293 cell-line was isolated which expressed high-levels of functional tTa. The tTa-producing cell-line was subsequently utilised in an attempt to isolate cell-lines which inducibly produce N-terminally Pk-tagged HN. These cells are currently undergoing selection for drug resistance. Further experiments were performed to try and develop a system whereby the low copy number of episomally-maintained EBV-based vectors present in a stable cell-line could be amplified to high copy number, whereby a subsequent increase in protein production would be envisaged. For this purpose, SV40 ori-containing, EBV-based vectors were constructed in which the expression of the non-toxic SV5 P protein was under the control of the hCMV IE promoter/enhancer. Cell-lines were isolated which produced the SV5 P in various amounts. Transient amplification of the episomal copy number was attempted via a transient expression of the SV40 LTAg, using plasmid DNA transfection. No subsequent increase in protein production was observed by way of a Western blot analysis.
Thesis, PhD Doctor of Philosophy
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