Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.
Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.
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重组大肠杆菌中产生的远缘链球菌葡聚糖酶的纯化和表征以及葡聚糖酶基因的序列分析。
DOI：
10.1128/jb.176.13.3839-3850.1994
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发表时间：
1994
影响因子：
3.2
通讯作者：
Curtiss3rd,R
中科院分区：
文献类型：
--
作者：
Wanda,SY;Curtiss3rd,R
携带远缘链球菌UAB66(g型)葡聚糖酶(Dex)基因的质粒(PYA902)产生一种C端截短的葡聚糖酶(Dex)，其质量形式多样，大小在80~130 kDa之间。对大肠杆菌产生的酶进行了纯化和鉴定，并在兔体内产生了抗体。纯化的葡聚糖酶的天然相对分子质量为160~260 kDa，比活力为4000U/mg蛋白质。用不同的抗血清和免疫印迹分析，研究了不同重组克隆所特异性的葡聚糖酶与SPAA蛋白之间潜在的免疫交叉反应。未观察到交叉反应。最适pH(5.3)、最适温度(39℃)和等电点(3.56、3.6、3.7)与从远缘链霉菌中纯化的葡聚糖酶相似。确定了Dex DNA限制性内切酶图谱，获得了多个亚克隆。利用染色体DNA亚克隆pYA993、pYA3009和UAB66测定了Dex基因的核苷酸序列。Dex的开放阅读框为4,011个碱基对，以终止密码子TAA结尾。在起始密码子之前发现了一个核糖体结合位点和可能的启动子。推导的氨基酸序列显示存在一个由30个氨基酸组成的信号肽。通过对E.Colichi 2831(PYA902)产生的Dex的N端氨基酸序列分析，确定了信号序列的切割位点。C末端由富含丝氨酸和苏氨酸的多肽LPKTGD、3个带电荷的氨基酸、19个具有强烈疏水性的氨基酸和一个带电的五肽尾部组成，它们被认为对应于革兰氏阳性球菌的细胞壁跨越区、LPXTGX共同序列和表面相关蛋白的膜锚定结构域。
The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.