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.

中文翻译：

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使用内含肽介导的延迟蛋白自动激活在酵母中表达高活性细菌磷脂酶 A2。

磷脂酶 A2 (PLA2) 已在工业中得到广泛应用。然而，由于其对细胞膜的毒性，在不同表达系统中产生重组 PLA2 是一项艰巨的任务。我们在此报告开发了一种从酵母酿酒酵母中的紫色链霉菌 A-2688 菌株中生产高活性 PLA2 的有效方法。该方法基于使用插入到磷脂酶序列中的 PRP8 小内含肽（来自 Penicillium chrysogenum），目的是使酶暂时失活并随后延迟自激活。我们证明最有效的内含肽插入位点是成熟磷脂酶的 Ser76。由于酵母细胞分泌含有内含肽的前体及其随后的自催化剪接，酵母培养液中积累的高活性酶。所得重组磷脂酶A2蛋白的性质与天然紫罗兰链霉菌PLA2蛋白的性质相似。还讨论了含内含蛋白的延迟自激活的可能进化作用。