Abstract
This manuscript describes cloning, production, refolding, and autocleavage of Pcal_0970, a plant-type L-asparaginase from hyperthermophilic archaeon Pyrobaculum calidifontis. Co-expression of the gene encoding Pcal_0970 with molecular chaperon GroEL from Geobacillus thermopakistaniensis, resulted in production of soluble but heat labile recombinant Pcal_0970. The insoluble and inactive protein was solubilized and refolded in vitro by fractional dialysis. The refolded protein underwent complete autocleavage at 80 °C within 48 h, while the cleavage rate was significantly slow at lower temperatures. Free glycine, pH, and metal ions did not have any effect on autocleavage of Pcal_0970. Structural analysis showed the absence of K+ binding conserved sequence in Pcal_0970, predicting it to be a K+-independent L-asparaginase. To the best of our knowledge, this is the first report on temperature dependent autocleavage and activation of a highly thermostable plant-type L-asparaginase from phylum Crenarchaeota.
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This work was partially supported by Higher Education Commission, Pakistan through Grant No. CPEC-24 to NR.
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Sajed, M., Falak, S., Muhammad, M.A. et al. A plant-type L-asparaginase from Pyrobaculum calidifontis undergoes temperature dependent autocleavage. Biologia 77, 3623–3631 (2022). https://doi.org/10.1007/s11756-022-01215-3
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DOI: https://doi.org/10.1007/s11756-022-01215-3