{"id":18392,"date":"2026-08-24T14:43:13","date_gmt":"2026-08-24T14:43:13","guid":{"rendered":"https:\/\/www.sygnaturediscovery.com\/?post_type=case-study&p=18392"},"modified":"2026-08-24T15:07:11","modified_gmt":"2026-08-24T15:07:11","slug":"generating-a-crystallography-ready-secreted-protein-through-construct-and-glycosylation-optimisation","status":"publish","type":"case-study","link":"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/generating-a-crystallography-ready-secreted-protein-through-construct-and-glycosylation-optimisation\/","title":{"rendered":"Generating a Crystallography-Ready Secreted Protein Through Construct and Glycosylation Optimisation"},"content":{"rendered":"\n

The Challenge<\/strong><\/h2>\n\n\n\n

A client required more than 2 mg of a novel secreted protein to support X-ray crystallography<\/a> studies.<\/p>\n\n\n\n

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.<\/p>\n\n\n\n

Our Approach<\/strong><\/h2>\n\n\n\n

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.<\/p>\n\n\n\n

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.<\/p>\n\n\n\n

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.<\/p>\n\n\n\n

This approach produced protein with a purity greater than 95%.<\/p>\n\n\n\n

Characterizing Protein Heterogeneity<\/strong><\/h3>\n\n\n\n

Following purification, the protein was analysed using SDS-PAGE and mass spectrometry.<\/p>\n\n\n\n

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.<\/p>\n\n\n\n

The low level of occupancy suggested that N-linked glycosylation was not required for correct protein folding.<\/p>\n\n\n\n

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.<\/p>\n\n\n\n

Expression and purification of the modified construct proceeded in a similar manner to the original protein.<\/p>\n\n\n\n

Supporting Construct Selection Through Mass Spectrometry<\/strong><\/h3>\n\n\n\n

Mass spectrometry was used to characterise the engineered protein construct and to assess the impact of removing the N-linked glycosylation site.<\/p>\n\n\n\n

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

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.<\/p>\n\n\n\n

Outcome<\/strong><\/h2>\n\n\n\n

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.<\/p>\n\n\n\n

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

Why It Matters<\/strong><\/h2>\n\n\n\n

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.<\/p>\n\n\n\n

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

Key Results<\/strong><\/h2>\n\n\n\n

\u2705 Three structurally guided protein constructs evaluated<\/p>\n\n\n\n

\u2705 Secreted expression achieved in HEK cells<\/p>\n\n\n\n

\u2705 Expression levels of 5-10 mg\/L obtained for the highest-performing constructs<\/p>\n\n\n\n

\u2705 Greater than 95% purity achieved using cation exchange and size exclusion chromatography<\/p>\n\n\n\n

\u2705 N-linked glycosylation heterogeneity identified and characterised<\/p>\n\n\n\n

\u2705 Alternative construct generated based on analytical findings<\/p>\n\n\n\n

\u2705 Protein supplied for crystallisation studies<\/p>\n\n\n\n

\u2705 Crystals suitable for X-ray diffraction successfully obtained<\/p>\n\n\n\n

<\/p>\n","protected":false},"excerpt":{"rendered":"","protected":false},"featured_media":0,"template":"","category":[770,680,703,766,702],"resource_tag":[],"class_list":["post-18392","case-study","type-case-study","status-publish","hentry","category-crystallography","category-protein-and-structure","category-protein-characterisation","category-protein-expression","category-protein-expression-and-purification"],"acf":[],"yoast_head":"\nGenerating a Crystallography-Ready Secreted Protein<\/title>\n<meta name=\"description\" content=\"How expression, purification and protein characterization enabled the production of a cystallography-ready protein.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/generating-a-crystallography-ready-secreted-protein-through-construct-and-glycosylation-optimisation\/\" \/>\n<meta property=\"og:locale\" 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