Okamoto K, Nomura T, Hamada M, Fukuda T, Noguchi Y, Fujii Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan.
Microbiol Immunol. 2000;44(9):787-98. doi: 10.1111/j.1348-0421.2000.tb02565.x.
We cloned a protease gene of Aeromonas sobria and determined its nucleotide sequence. The protease is composed of 624 amino acid residues and its calculated molecular weight is 66,737.7. The amino acid sequence showed the characteristic features of a bacterial serine protease. We expressed the protease gene in Vibrio parahaemolyticus from which the synthesized protease is secreted into the culture medium as the mature form, and purified the mature protease by successive column chromatographies. The size of the mature protease is 65,000 daltons and the amino acid sequence analysis revealed that a 24-amino acid peptide at the amino terminal of the precursor is removed from the mature protease. This peptide might function as a signal peptide in translocation across the inner membrane. Subsequently, we found that the protein, designated ORF2 protein, encoded by the gene lying adjacent to the 3' end of the protease gene plays an important role in production of the protease. Mutation of the ORF2 gene did not affect transcription of the protease gene, but resulted in degradation of the protease in the cell. This shows that ORF2 protein is required for the successful production of the serine protease by cell.
我们克隆了温和气单胞菌的一个蛋白酶基因，并测定了其核苷酸序列。该蛋白酶由624个氨基酸残基组成，其计算分子量为66,737.7。氨基酸序列显示出细菌丝氨酸蛋白酶的特征。我们在副溶血性弧菌中表达了该蛋白酶基因，合成的蛋白酶以成熟形式分泌到培养基中，并通过连续柱色谱法纯化了成熟蛋白酶。成熟蛋白酶的大小为65,000道尔顿，氨基酸序列分析表明，前体氨基末端的一个24个氨基酸的肽段从成熟蛋白酶中去除。该肽段可能在跨内膜转运中起信号肽的作用。随后，我们发现由位于蛋白酶基因3'端相邻的基因编码的蛋白质，命名为ORF2蛋白，在蛋白酶的产生中起重要作用。ORF2基因的突变不影响蛋白酶基因的转录，但导致细胞内蛋白酶的降解。这表明ORF2蛋白是细胞成功产生丝氨酸蛋白酶所必需的。
