BACKGROUND The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. RESULTS We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. CONCLUSION Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.

中文翻译：

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胞外链霉菌中Aeropyrum pernix K1产生工程热稳定蛋白酶pernisine的细胞外生产。

背景技术热稳定的丝氨酸蛋白酶pernisine源于嗜热古生气生孢菌pernix，并具有有价值的工业应用。由于其特性，无法在标准的工业发酵设施中种植多年生曲霉。此外，由于其活化涉及相对复杂的加工步骤，因此鸟氨酸是在嗜温异源宿主中异源表达的高要求靶标。结果我们通过应用逐步蛋白质工程方法，通过密码子优化基因的异源表达，在链霉菌链球菌宿主中产生了活性细胞外鸟氨酸。为了确保分泌完全活性的酶，将来自rimusus s.rimosus蛋白酶的srT信号序列与pernisine融合。为了促进正确的精氨酸加工和折叠，将srT功能性切割位点基序直接融合到核心鸟氨酸序列上，从而省去了前区。对野生型和重组鸟氨酸的比较生化分析证实，由棉铃虫产生的酶保留了天然鸟氨酸的所有所需特性。重要的是，重组鸟氨酸还降解了细胞和感染性牛病毒蛋白，这是该蛋白酶的特殊应用之一。结论在嗜热链球菌异源宿主中成功生产了保留嗜热宿主天然酶所有有利特性的功能性鸟氨酸。重要的是，我们实现了细胞内活性鸟氨酸的生产，这大大简化了进一步的下游程序，并且省去了对其激活进行任何预处理步骤的需要。我们证明，S。rimosus可以用作工业嗜热生物的重组蛋白的工业生产的有吸引力的主机。