| Summary: | <p>Abstract</p> <p>Background</p> <p>2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.</p> <p>Findings</p> <p>In order to be able to fully characterize CNPase structurally and functionally, we have set up expression systems for different domains of CNPase, using a total of 18 different expression constructs. CNPase was expressed in <it>E. coli </it>with a TEV-cleavable His-tag. Enzymatic activity assays indicated that the purified proteins were active and correctly folded. The folding of both the full-length protein, as well as the N- and C-terminal domains, was also studied by synchrotron CD spectroscopy. A thermal shift assay was used to optimize buffer compositions to be used during purification and storage. The assay also indicated that CNPase was most stable at a pH of 5.5, and could be significantly stabilized by high salt concentrations.</p> <p>Conclusions</p> <p>We have been able to express and purify recombinantly several different domains of CNPase, including the isolated N-terminal domain, which is folded mainly into a β-sheet structure. The expression system can be used as an efficient tool to elucidate the role of CNPase in the myelin sheath.</p>
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