Future experiments

In order to obtain the native fusion protein, the purification protocol has to be optimised. Future purifications will include protease inhibitors that might prevent the formation of truncated proteins. To increase the binding affinity of the fusion protein to the nickel resin, imidazole concentration and pH of the binding buffer have to be optimised. A rearrangement of the expression vector that produces the protein Lstag-sfc-10-TEVcs-MBP-His might lead to the production of a protein that can be purified with IMAC (Fig. 6.1B). In this scenario, Lstag-sfc-10 is located at the N-terminus of the protein, followed by a TEV cleavage site and the solubilising MBP, which is His-tagged at the C-terminus. Potentially, the His-tag will not be hidden by the tertiary structure and is fully accessible for resin binding. Another approach to produce soluble protein involves expression in a eukaryotic expression hosts such as insect cells [37] or the yeasts Pichia pastoris [38] and Saccharomyces cerevisiae [39]. If it is not possible to produce the target protein in the soluble form, calcification assays can be performed with the insoluble protein as produced from the pET16b+Lstag-sfc-10-His (Fig. 6.2B). Previously, a biomineralising protein that was purified under denaturing conditions and refolded in vitro was successfully deployed in an calcification assays [20].

An optimised protocol that allows the production of Lstag-sfc-10 in the heterologous expression host E. coli will be useful to provide sufficient protein for various protein assays, that will help elucidate the role of the protein in the shell forming process.

Supplementary files

The supplementary files can be found in the appendix 2.

Supplementary S. 6.1: List of all primers used in this study Supplementary S. 6.2: Insert and protein sequences

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