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首页> 外文期刊>Journal of bacteriology >Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.
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Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

机译：平肠肠球菌muramidase-2基因的克隆和序列分析。

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Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.
机译：通过底物结合，胍盐酸盐萃取和反相色谱将平肠肠球菌ATCC 9790的胞外muramidase-2纯化至均质。使用特异性识别muramidase-2的单克隆抗体2F8来筛选λ噬菌体gt11中的E. hirae ATCC 9790 DNA基因组文库。分离并分析了含有4.5kb DNA插入片段的阳性噬菌体克隆。使用限制酶KpnI，Sau3AI和PstI将EcoRI消化的4.5-kb片段切成2.3-，1.0-和1.5-kb片段，并将每个片段亚克隆到质粒pJDC9或pUC19中。确定每个亚克隆的核苷酸序列。序列数据显示了一个开放阅读框，其编码一个666个氨基酸残基的多肽，计算分子量为70,678 Da。纯化的胞外muramidase-2的前24个N末端氨基酸与推定的49个氨基酸的信号序列后的氨基酸序列非常吻合。对推导的氨基酸序列的分析表明，在蛋白质的C-末端区域存在六个高度同源的重复单元，这些重复单元被高度富含丝氨酸和苏氨酸的非同源中间序列隔开。整个序列与最近克隆的粪便链球菌自溶素具有高度同源性。

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