Three extracellular proteases from Cochliobolus carbonum: cloning and targeted disruption of ALP1.
Three extracellular proteases from Cochliobolus carbonum: cloning and targeted disruption of ALP1.
复制标题
来自 Cochliobolus Carbonum 的三种胞外蛋白酶：ALP1 的克隆和靶向破坏。
DOI：
10.1094/mpmi-9-0290
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Walton,JD
中科院分区：
文献类型：
--
作者：
Murphy,JM;Walton,JD
分离纯化了三种细胞外丝氨酸蛋白酶（Alp1a, Alp1b, Alp2）。在8种碳/蛋白基质中，当真菌生长在含有胶原蛋白的培养基上时，总蛋白酶活性最高。Alp1a和Alp1b是胰蛋白酶家族的成员（EC 3.4）。21.4)， Alp2是枯草菌素家族的成员（EC 3.4）。21.62)。Alp1a、Alp1b和Alp2的单体分子质量分别为25、30和38 kDa。Alp1b是糖基化的，而Alp1a不是。编码Alp1a的基因ALP1是基于两个氨基酸序列的PCR引物分离得到的：一个直接从Alp1a的n端获得，另一个在其他胰蛋白酶中高度保守。用RACE确定转录起始位点，用cDNA克隆确定内含子结构和多聚腺苷化位点。利用ALP1的内部片段通过转化介导的基因破坏创建Alp1a零突变体。突变体的总蛋白酶活性降低了35% ~ 45%。通过色谱分析，突变体失去了两个紫外吸收峰和Alp1a和Alp1b对应的两个蛋白酶活性，结合生化数据表明，Alp1a和Alp1b是同一基因的产物。ALP1突变体的体外生长和疾病表型与野生型菌株有明显区别；因此，ALP1本身并不是致病性所必需的。
Three extracellular serine proteases (Alp1a, Alp1b, Alp2) from Cochliobolus carbonum were purified and characterized. Of eight carbon/protein substrates tested, total protease activity was highest when the fungus was grown on medium containing collagen. Alp1a and Alp1b are members of the trypsin family (EC 3.4. 21.4), and Alp2 is a member of the subtilisin family (EC 3.4. 21.62). Alp1a, Alp1b, and Alp2 have monomer molecular masses of 25, 30, and 38 kDa respectively. Alp1b is glycosylated, whereas Alp1a is not. The gene encoding Alp1a, ALP1, was isolated using PCR primers based on two amino acid sequences: One obtained directly from the N-terminus of Alp1a and another that is highly conserved in other trypsins. The transcriptional start site was determined using RACE and the intron structure and polyadenylation site were determined from a cDNA clone. An internal fragment of ALP1 was used to create Alp1a null mutants by transformation-mediated gene disruption. Total protease activity in the mutants was reduced by 35% to 45%. By chromatographic analysis, the mutants had lost two peaks of UV absorption and the two protease activities corresponding to Alp1a and Alp1b, which, together with the biochemical data, indicates that Alp1a and Alp1b are products of the same gene. The in vitro growth and diseases phenotypes of the ALP1 mutants were distinguishable from the wild-type strain; therefore, ALP1 is not by itself required for pathogenicity.