Abstract
N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in Escherichia coli, in particular in proteins bearing an N-terminal histidine-tag. This post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of 13C/15N labeled proteins, their chemical shifts were not yet reported. Here we present the complete 1H and 13C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His)6. In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.
Highlights
Post-translational protein modifications (PTMs) add another layer of complexity to the proteome and each modification can potentially change the structure, function and stability of a protein (Aebersold et al 2018)
Chemical shift correlations of the gluconoyl group In NMR spectra of recombinant Coprinopsis cinerea lectin 2 (CCL2) in a 13C/15N labeled form (Schubert et al 2012) we observed quite intense and sharp 1H–13C chemical shift correlations with 13C chemical shifts between 70 and 78 ppm (Fig. 2), and since it was not possible to get rid of those signals by changing the purification scheme, we considered that these signals arose from a post-translational modification
The degree of gluconoylation of certain recombinant proteins expressed in E. coli depends on the type of medium
Summary
Post-translational protein modifications (PTMs) add another layer of complexity to the proteome and each modification can potentially change the structure, function and stability of a protein (Aebersold et al 2018). Knowledge about the protein homogeneity and the presence of PTMs is crucial for most applications, e.g. pharmaceutical applications. N-terminal gluconoylation and phosphogluconoylation were first noticed by mass spectrometry in recombinant proteins expressed in Escherichia coli (Geoghegan et al 1999; Yan et al 1999a). The phosphate group is typically cleaved by host cell phosphatases leading to gluconoyl. The occurrence of these modifications depends on the N-terminal sequence, and likely on the bacterial strain and the bacterial growth conditions. BL21(DE3), the most commonly used E. coli strain for protein expression, is known to accumulate 6-phosphogluconolactone due to the lack of 6-phosphogluconolactonase (Meier et al 2012), which favors gluconoylation, so that it is not unexpected that this strain produces significant
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