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Molecular cloning and expression of genes encoding proteolytic enzymes

机译:编码蛋白水解酶的基因的分子克隆和表达

摘要

A novel expression cassette comprises, in the direction of transcription, a transcriptional regulatory region and a translational initiation region functional in a Bacillus host cell, a DNA sequence encoding a high alkaline proteolytic enzyme and translational and transcriptional termination regions functional in the host cell, where expression of the DNA sequence is under regulatory control of the initiation and termination regions, and at least one of a marker gene and a temp.-sensitive origin of replication of a bacterial plasmid. Also claimed is a method for transforming alkalophilic Bacillus cells which comprises: (a) pretreating the Bacillus cells with lysozyme in an alkaline medium at 20-37 deg.C to form protoplasts, (b) sepg. the protoplasts from the lysozyme, (c) combining the protoplasts with a DNA construct in the presence of a fusogen at 20-37 deg.C, (d) diluting the fusogen and regenerating the Bacillus cells and (e) growing the regenerated Bacillus cells under selective conditions. Also claimed is a DNA sequence encoding a serine protease originating from Bacillus novo species PB92. Also claimed is an isolated DNA or RNA oligonucleotide comprising at least 15 sequential nucleotides capable of hybridising with a sequence that encodes an amino acid sequence of a high alkaline serine protease obtainable from Bacillus novo species PB92.
机译:新颖的表达盒在转录方向上包括在芽孢杆菌属宿主细胞中起作用的转录调节区和翻译起始区,编码高碱性蛋白水解酶的DNA序列以及在宿主细胞中起作用的翻译和转录终止区。 DNA序列的表达在起始和终止区以及标记基因和温度敏感的细菌质粒复制起点中的至少一个的调控下。还要求保护的是一种转化嗜碱芽孢杆菌细胞的方法,该方法包括:(a)在20-37℃的碱性介质中用溶菌酶预处理芽孢杆菌细胞以形成原生质体,(b)sepg。来自溶菌酶的原生质体,(c)在20-37℃下在融合剂存在下将原生质体与DNA构建体结合,(d)稀释融合素并再生芽孢杆菌细胞,以及(e)使再生的芽孢杆菌细胞生长在选择性条件下。还要求保护编码源自新芽孢杆菌种PB92的丝氨酸蛋白酶的DNA序列。还要求保护的是包含至少15个连续核苷酸的分离的DNA或RNA寡核苷酸,其能够与编码可得自新芽孢杆菌种PB92的高碱性丝氨酸蛋白酶的氨基酸序列的序列杂交。

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