Project Description

Generation of a novel, secreted protein for structure determination

Our client required >2 mg of a novel, secreted protein for X-ray structure determination studies. The protein was thought to be N-glycosylated.

Our challenge was to obtain sufficient >95% pure protein for crystallisation experiments, as measured by SDS-PAGE, size exclusion and mass spectrometry analysis.  In addition, we were also aiming to take forward the shortest, most stable construct feasible to maximise our chances of success in obtaining diffracting crystals for structure determination.

Our Solutions

Our solution was to evaluate three C-terminally deleted constructs for secreted expression in HEK cells, based on structural alignments with related proteins. Proteins were successfully secreted into the medium after transient transfection, with two constructs giving good yields of  expressed protein (5-10mg/L).
No protein purification tag was used as the protein had a very basic isoelectric point (pI) enabling purification via cation exchange and could be purified to >95% using a combination of cation exchange and size exclusion chromatography.
Analysis by SDS-PAGE and mass spectrometry demonstrated a heterogeneous product with respect to glycosylation. This was due to a single N-linked consensus (NXS/T) that was occupied in only 10-20% of the molecules. This suggested that glycosylation was not required for protein folding so a new construct and protein was generated where the Asn (N) was mutated to Asp (D), with expression and purification very similar to the non mutated construct. Mass spectrometry analysis showed the protein had a mass of 948Da higher than expected which would be consistent with a common O-linked tetrasaccharide structure.

The Impact

As a result of carefully evaluating the most suitable protein for crystallisation studies, we were able to produce the required amount of well characterised protein to generate crystals suitable for X-ray diffraction studies.

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