Maciver B, McHale R H, Saul D J, Bergquist P L
Centre for Gene Technology, University of Auckland, New Zealand.
Appl Environ Microbiol. 1994 Nov;60(11):3981-8. doi: 10.1128/aem.60.11.3981-3988.1994.
The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.
通过聚合酶链反应（PCR）扩增，鉴定出一种嗜热芽孢杆菌属丝氨酸蛋白酶的基因。在使用PCR产物作为探针，从Southern印迹中鉴定并分离出合适大小的限制性片段后，克隆了完整基因。还获得了另外两种不同的PCR产物，它们被证明源自嗜热芽孢杆菌属中存在的其他丝氨酸蛋白酶基因。序列分析显示一个1206 bp的开放阅读框，编码一个由401个氨基酸组成的多肽。该多肽被确定为一种具有信号序列和前序列的细胞外丝氨酸蛋白酶。成熟蛋白酶与多种芽孢杆菌属的枯草杆菌蛋白酶样丝氨酸蛋白酶具有同源性，与来自普通嗜热放线菌的丝氨酸蛋白酶嗜热菌蛋白酶有61%的同源性。该基因在表达载体pJLA602中的大肠杆菌中表达，并在克隆载体pGEM5中与lacZ基因的α肽融合表达。来自lacZ融合质粒的重组蛋白酶用于确定该酶的一些特性，其最适pH为8.5，最适温度为75℃，热稳定性范围为在90℃下半衰期为12.2分钟至在75℃下半衰期为40.3小时。该酶与杆菌肽柱结合，这种方法为生产该蛋白酶提供了一种简单的一步法，纯化至接近均一。
