Generating a Crystallography-Ready Secreted Protein Through Construct and Glycosylation Optimisation

The Challenge

A client required more than 2 mg of a novel secreted protein to support X-ray crystallography studies.

The target protein was predicted to contain N-linked glycosylation, introducing the potential for heterogeneity that could complicate structural biology workflows. The objective was therefore not simply to express the protein, but to identify a construct and production strategy capable of generating well-characterized material suitable for crystallisation experiments.

Our Approach

To increase the likelihood of obtaining a suitable protein for structural studies, three C-terminal deletion constructs were designed based on structural alignments with related proteins.

All three constructs were evaluated for secreted expression in HEK cells following transient transfection. Protein was successfully secreted into the culture medium, with two constructs producing expression levels of approximately 5-10 mg/L.

Rather than incorporating an affinity purification tag, the purification strategy was developed around the biochemical properties of the target protein. The protein possessed a highly basic isoelectric point (pI), allowing purification using cation exchange chromatography followed by size exclusion chromatography.

This approach produced protein with a purity greater than 95%.

Characterizing Protein Heterogeneity

Following purification, the protein was analysed using SDS-PAGE and mass spectrometry.

These studies revealed that the protein preparation was heterogeneous with respect to glycosylation. Further analysis identified a single N-linked glycosylation consensus sequence that was occupied in only 10-20% of protein molecules.

The low level of occupancy suggested that N-linked glycosylation was not required for correct protein folding.

Based on these findings, a modified construct was generated in which the asparagine residue associated with the N-linked glycosylation site was replaced with aspartic acid.

Expression and purification of the modified construct proceeded in a similar manner to the original protein.

Supporting Construct Selection Through Mass Spectrometry

Mass spectrometry was used to characterise the engineered protein construct and to assess the impact of removing the N-linked glycosylation site.

Analysis showed that the protein mass remained 948 Da higher than expected, consistent with the presence of a common O-linked tetrasaccharide structure.

These data provided additional insight into the post-translational modifications present within the protein preparation and contributed to selection of the most appropriate construct for crystallisation studies.

Outcome

By evaluating multiple construct designs and combining expression, purification, and protein characterisation data, Sygnature Discovery successfully produced the quantity of protein required for the client’s structural biology programme.

The resulting protein was sufficiently well characterised to support crystallisation studies and ultimately enabled the generation of crystals suitable for X-ray diffraction experiments.

Why It Matters

Producing proteins for structural biology often requires more than successful expression alone. Construct design, purification strategy, and characterisation of post-translational modifications can all influence whether a protein progresses successfully into crystallisation studies.

This project demonstrates how combining protein production expertise with analytical characterisation can help identify the most suitable protein construct for downstream structural biology applications.

Key Results

✅ Three structurally guided protein constructs evaluated

✅ Secreted expression achieved in HEK cells

✅ Expression levels of 5-10 mg/L obtained for the highest-performing constructs

✅ Greater than 95% purity achieved using cation exchange and size exclusion chromatography

✅ N-linked glycosylation heterogeneity identified and characterised

✅ Alternative construct generated based on analytical findings

✅ Protein supplied for crystallisation studies

✅ Crystals suitable for X-ray diffraction successfully obtained