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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.
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Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.

机译：用于在细菌中高效表达哺乳动物蛋白的通用载体的构建：合成核糖体结合位点的使用。

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With the premise that mRNAs transcribed in Escherichia coli from cloned eukaryotic DNA inserts do not possess the necessary regulatory signals for recognition by prokaryotic ribosomes, we have constructed a general plasmid vector carrying a chemically synthesized prokaryotic ribosome binding site that will ensure the efficient expression of eukaryotic proteins in E. coli. In addition to the regulatory signals necessary for ribosome recognition, the synthetic segment contains, at one end, a Pst I cleavage site which will direct its insertion to pBR322 DNA and, at the other end, a HindIII site to facilitate attachment of the passenger eukaryotic gene. Using simian virus 40 (SV40) tumor (t) antigen as a model system, we have ligated the SV40 DNA fragment containing the entire t antigen gene in tandem with the synthetic ribosome binding site to pBR322 DNA at the Pst I site, which lies within the coding sequence of the beta-lactamase gene. Initiation of transcription at the beta-lactamase promoter would produce a chimeric mRNA with the synthetic ribosome binding signals and the SV40 sequence flanked by beta-lactamase coding sequences. Utilization of the synthetic regulatory signals for initiation of translation is demonstrated by the efficient synthesis, in bacterial transformants, of authentic SV40 t antigen. Excision of the entire SV40 insert by HindIII from those clones that have retained intact HindIII sites at the junction between the ribosome binding site and the SV40 sequence would allow insertion of other heterologous DNAs by using HindIII linkers. The efficient expression of any DNA insert would require that the entire coding sequence be contiguous and that its termini be randomized by treatment with exonuclease III and nuclease S1 to vary the distance between the translational initiation codon and the synthetic ribosome binding site.
机译：前提是从克隆的真核生物DNA插入物中转录的mRNA不具有原核生物核糖体识别所必需的调控信号，因此我们构建了带有化学合成的原核生物核糖体结合位点的通用质粒载体，该质粒将确保真核生物的有效表达大肠杆菌中的蛋白质。除了核糖体识别所必需的调控信号外，合成片段的一端还包含一个Pst I切割位点，该位点将引导其插入pBR322 DNA，另一端包含一个HindIII位点，以利于乘客真核生物的附着。基因。使用猿猴病毒40（SV40）肿瘤（t）抗原作为模型系统，我们将包含完整t抗原基因的SV40 DNA片段与位于Pst I位点的pBR322 DNA的合成核糖体结合位点串联在一起β-内酰胺酶基因的编码序列。在β-内酰胺酶启动子处转录的起始将产生具有合成核糖体结合信号和侧接于β-内酰胺酶编码序列的SV40序列的嵌合mRNA。通过在细菌转化体中有效合成真实的SV40 t抗原，证明了利用合成的调控信号进行翻译。从那些在核糖体结合位点和SV40序列之间的连接处保留了完整的HindIII位点的克隆中，用HindIII切除整个SV40插入片段将允许使用HindIII连接子插入其他异源DNA。任何DNA插入片段的有效表达都要求整个编码序列是连续的，并且其末端要通过用核酸外切酶III和核酸酶S1处理来随机化，以改变翻译起始密码子与合成核糖体结合位点之间的距离。

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