Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli.
Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli.
复制标题
使用大肠杆菌脂蛋白基因构建多功能表达克隆载体。
DOI：
10.1002/j.1460-2075.1982.tb01244.x
复制
发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Inouye,M
中科院分区：
文献类型：
--
作者：
Nakamura,K;Inouye,M
外膜脂蛋白（lpp）基因是大肠杆菌中最丰富的蛋白质，用于构建多用途表达克隆载体。就基因表达而言，这些载体由两种类型组成，一种用于组成型（pIN-I型;三种载体），另一种用于诱导型（pIN-II型;三种载体）基因表达，并且具有以下特征：（a）将lpp基因插入多拷贝质粒pBR 322中，并且去除泰特基因以保持载体的大小最小（约5 kb）。(b)将含有EcoRI、HindIII和BamHI位点的22 bp核苷酸序列插入前脂蛋白的第三个氨基酸的位置。(c)相同的核苷酸序列也被插入在相同位置的另外两个阅读框中。车辆中没有其他EcoRI、HindIII和BamHI位点。因此，六种不同类型的限制性片段（EcoRI-EcoRI、HindIII-HindIII、BamHI-BamHI、EcoRI-HindIII、EcoRI-BamHI和HindIII-BamHI）可以在三种不同阅读框中的任何一种中的该位置克隆。(d)缺失lpp基因第46 - 168位的核苷酸序列。而lpp基因的3′端154 bp的位置被保留下来，不仅包含了3个不同阅读框的翻译终止密码子，而且还包含了lpp基因的转录终止信号。因此，该序列被认为是为了防止不必要的翻译以及克隆基因的转录通读。（250字处删节）
The gene for the outer membrane lipoprotein (lpp), the most abundant protein of Escherichia coli, was used to construct multi‐purpose expression cloning vehicles. These vehicles consist of two types in terms of gene expression, one for constitutive (pIN‐I type; three vehicles) and the other for inducible (pIN‐II type; three vehicles) gene expression, and have the following features: (a) The lpp gene was inserted into a multicopy plasmid, pBR322, and the tet gene was removed to keep the size of the vehicles minimal (approximately 5 kb). (b) A nucleotide sequence of 22 bp which contains EcoRI, HindIII, and BamHI sites was inserted at the position of the third amino acid of the prolipoprotein. (c) The same nucleotide sequence was also inserted in two other reading frames at the same position. There are no other EcoRI, HindIII, and BamHI sites in the vehicles. Therefore, six different types of restriction fragments (EcoRI‐EcoRI, HindIII‐HindIII, BamHI‐BamHI, EcoRI‐HindIII, EcoRI‐BamHI, and HindIII‐BamHI) can be cloned at this position in any of the three different reading frames. (d) The nucleotide sequence from position 46 to 168 of the lpp gene was deleted. However, the 3′ end position of the lpp gene of 154 bp was retained, which contains not only translation termination codons in three different reading frames but also the transcription termination signal of the lpp gene. Thus, this sequence is assumed to prevent unnecessary translation as well as transcriptional read‐through of a cloned gene.(ABSTRACT TRUNCATED AT 250 WORDS)