Presentation of a new method of producing big Insulin-like Growth Factor 2, validation of this method and research into a new antibody-free quantification method for Insulin-like growth factor 2

Abstract

Expression of Insulin-like Growth Factor 2 (IGF2) is notoriously challenging, especially the elusive non-glycosylated big IGF2⁸⁷. Recent advances have shown that mature IGF2 can be stabilised by the inclusion of C-domain mutations N29 and S39_PQ which decrease the conformational space occupied by the protein (Hexnerová et al., J. Biol. Chem., 2016, 291, 21234– 21245). The original refolding and purification technique presented by Hexnerová et al. was modified by use of new expression plasmids and processing techniques. This new protocol produces folded protein in good yields.

The folded state of the mature IGF2 isoforms produced was probed by 1D ¹H NMR, and these results were validated by binding of the IGF2 mutants as shown by NMR upon their titration into ¹⁵N isotopically enriched Domain 11ᴬᴮ³ at pH 4.0, which served as a useful probe for this interaction. The insolubility of C-domain mutant big IGF2 at low pH and of Domain 11ᴬᴮ³ at elevated pH meant that this technique could not be used here. Domain 11ᴬᴮ³ was modified to include three C-terminal aspartic acid residues to lower its pI and increase solubility at elevated pH. NMR data was collected during titrations of C-domain mutant big IGF2 with this pI shifted Domain 11ᴬᴮ³ which showed only limited evidence of binding. However, a native mass spectrometry protocol was developed and successfully employed which showed that each IGF2 isoform produced binds to Domain 11ᴬᴮ³, but not to an IGF2-binding knockout Domain 11 mutant. This verifies that the IGF2 production, refolding and purification protocol can be used to produce IGF2⁸⁷.

Additionally, work was carried out to research two avenues for developing an antibody-free IGF2 assay. The first was a FRET-based competition assay. Fluorescent IGF2⁶⁷ was produced by reaction of protein with an NHS-ester fluorophore. Fluorescent Domain 11 was produced by producing a fluorophore-tagged CoA and enzymatically reacting this with a folded C-terminally ACP-S8 tagged Domain 11ᴬᴮ³. The second IGF2 quantification methodology developed was an Absolute Quantification (AQUA) mass spectrometry protocol, which compared trypsin-digested protein fragments to isotopically enriched standards by mass spectrometry. This shows promising early results using isotopically enriched IGF2 as a standard, though efforts to diversify this technique to enable multiplex analysis via use of concatenated proteins were shown to require further refinement. The results of these two IGF2 quantification assays may aid in the future development of an antibody free IGF2 quantification method.

Date of Award	23 Mar 2021
Original language	English
Awarding Institution	University of Bristol
Supervisor	Matthew P Crump (Supervisor) & Jens J W Holtvoeth (Supervisor)