Phospholipase A2 (PLA2) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A2 protein were similar to those of the native Streptomyces violaceoruber PLA2 protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.
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We thank F. Klebanov for the help with genetic engineering and Dr. S. Kuchin for the help with editing this manuscript.
This work was supported within the framework of the State Assignment no. 595-00003-19 PR.
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The authors declare that they have no conflict of interest.
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Cheperegin, S.E., Malysheva, A.V., Sannikova, E.P. et al. Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation. Appl Biochem Biotechnol 193, 1351–1364 (2021). https://doi.org/10.1007/s12010-020-03333-7
- Phospholipase A2 (PLA2)
- Streptomyces violaceoruber
- Saccharomyces cerevisiae