Understanding the Role of C-terminal Lysine in Intracellular Secretory Pathways

UoM administered thesis: Phd

  • Authors:
  • Mark Owen

Abstract

Improved understanding of factors which impact the expression of recombinant immunoglobulin (IgG)-based therapies can help to decrease manufacturing process costs and therefore bring down cost to patients. Unintended modifications to the C-terminal region of IgG therapies as a consequence of recombinant expression results in product heterogeneity. Such heterogeneity is associated with changes in product quality and efficacy. One source of C-terminal heterogeneity is inconsistent C-terminal lysine clipping. In an effort to remove this source of heterogeneity, different groups have generated IgG mutants with genetic deletion of the C-terminal lysine residue (C-Lys variants). While genetic removal of the C-terminal lysine did remove this source of heterogeneity, the recombinant expression of such proteins was significantly decreased. This thesis aimed to better characterise the relationship between C-terminal amino acid structures and extent of expression, with particular focus on the C-terminal of IgG molecules. An additional aim of the thesis was to better-characterise the cellular processes underpinning the differences in expression of the wildtype and C-Lys proteins. To achieve the above aims, this thesis used two broad approaches. The first was a structural approach which generated a series of point mutations at the C-terminal of model IgG proteins. In addition to mutating to the IgG C-terminal, fusion of IgG C-terminal sequences of differing lengths to a fluorescent mCherry reporter molecule was carried out. Using these model proteins, an assay investigated the effect of the C-termini on the extent of expression. The second approach used a range of cellular analyses including flow cytometry, intracellular protein extraction, protein co-localisation analysis and a cell-free expression system to characterise how C- Lys variants were handled and expressed differently within the cell compared to its wildtype counterparts. This thesis showed that a short sequence of specific C-terminal amino acids was able to modulate the extent of protein expression. With this understanding, different C-terminal motifs were generated which removed incomplete lysine clipping as a source of product heterogeneity, while also facilitating expression which was comparable to the wildtype. The usage of different C-terminal “tags” generated here may also be useful to control the level of expression of other protein targets. In addition, when analysing the relationship between C-terminal structure and extent of expression, this thesis showed that diminished expression of C-Lys variants was likely to be due to a slower rate of translation compared to proteins which were capped with a lysine residue. Potential mechanistic explanations for the differences in translation rate are proposed, and future experimental steps are discussed.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date31 Dec 2021