Abstract

Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90 °C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90 °C. These results underline the importance of the Ca²⁺ binding sites predicted in these outer regions for stability and activity of pernisine.

Key points

• Aggregation of produced pernisine was prevented by translocation into periplasm.

• Thermostability of mature pernisine was increased.

• The outer regions of the catalytic core are required for pernisine thermostability.

中文翻译：

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大肠杆菌中pernisine的周质产生及其高热稳定性的决定因素。

摘要

Pernisine是超嗜热古细菌Aeropyrum pernix分泌的一种枯草杆菌蛋白酶样丝氨酸蛋白酶。该蛋白酶的显着特性是卓越的稳定性和降解感染性病毒蛋白的能力。在这里，我们显示了大肠杆菌周质中pernisine的产生。该策略阻止了鸟氨酸在细胞质中的聚集，并提高了分离的鸟氨酸的纯度。与以前的研究相比，该重组鸟氨酸的热稳定性显着提高。此外，构建了数个截短的pernisine变体并在大肠杆菌中表达识别最小活动域。pernisine的催化结构域由侧接N端和C端外部区域的αẞα结构相似的核心组成。C端α螺旋的缺失不影响90°C时的鸟氨酸活性。但是，C末端外部区域的完全删除导致蛋白水解活性的损失。缺失了N末端外部区域的pernisine变体在90°C下的活性降低。这些结果强调了在这些外部区域中预测的Ca ²⁺结合位点对于pernisine的稳定性和活性的重要性。

关键点

•通过转移到周质中来防止产生的鸟氨酸的聚集。

•成熟的鸟氨酸的热稳定性得到提高。

•精氨酸的热稳定性需要催化核的外部区域。