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Production and analysis of an extensively post-translationally modified secreted protein2018-11-23T12:03:05+00:00

Project Description

Production and analysis of an extensively post-translationally modified secreted protein

We were asked to generate a secreted protein that is known to undergo significant post-translational modification. The challenge was to not only make it but to clearly demonstrate that all the required post-translational modifications had occurred.

Our Solutions

An HEK293 system was chosen because, as a mammalian cell line, this was most likely to generate the required modifications. The protein was secreted using an efficient, artificial signal sequence. It had a 6His tag to enable affinity purification and a >95% pure protein was achieved by following this with size exclusion chromatography. The protein needed to be cleaved at a furin cleavage site into two subunits (alpha and beta) that were held together by a single intermolecular disulphide bond. Finally, although not thought to be essential for folding and activity, it was of interest to determine the glycosylation status of the protein for
protein structure determination.

SDS-PAGE analysis of the purified product showed a single band under non-reducing conditions and somewhat unexpectedly three species when reduced, two of which had similar masses on the gel. These three species were all analysed by tryptic peptide mapping using a SCIEX X500B LC-MS/MS mass spectrometer.  This revealed that one of the bands was clearly the alpha subunit and the other two were both the beta subunit with one major difference. With the slightly smaller subunit, a peptide could be clearly identified containing the N-linked glycosylation consensus N-X-S/T. The same peptide in the other beta band was absent suggesting that it was glycosylated, hence the slightly increased mass.

The Impact

Using a combination of HEK cell expression and detailed analysis we were able to generate a pure protein and demonstrate that all the required post-translation modifications had occurred.

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