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基因表达：大肠杆菌核糖体结合位点的化学合成及其在指导细菌中哺乳动物蛋白表达中的应用。

Gene expression: chemical synthesis of E. coli ribosome binding sites and their use in directing the expression of mammalian proteins in bacteria.

作者信息

Jay E, Seth A K, Rommens J, Sood A, Jay G

出版信息

Nucleic Acids Res. 1982 Oct 25;10(20):6319-29. doi: 10.1093/nar/10.20.6319.

DOI:10.1093/nar/10.20.6319
PMID:6294598
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC326921/
Abstract

Mammalian genes, when inserted into bacterial plasmid or phage DNAs, will not be expressed into the corresponding specific proteins in E. coli unless proper initiations signals required for recognition by E. coli ribosomes are provided. We have studied these signals and chemically synthesized two DNA duplexes each containing different initiation signals. These have been inserted in front of the Simian virus 40 (SV40) small tumor antigen gene (SV40 t gene) at varying distances from the ATG initiation codon prior to its cloning into pBR322 plasmid DNA. Plasmid containing clones carrying either of these two synthetic ribosome binding sites (RBS) at varying distances from the SV40 t gene all produced a 17K protein identical to authentic t antigen by immunologic, electrophoretic and proteolytic digestion analyses. This provides a novel method to ensure the specific expression of any contiguous mammalian gene to be cloned to bacteria, and also a unique in vivo method for studying the structure-function (efficiency) relationship of RBS with specific base changes.

摘要

哺乳动物基因插入细菌质粒或噬菌体DNA后，除非提供大肠杆菌核糖体识别所需的适当起始信号，否则在大肠杆菌中不会表达为相应的特定蛋白质。我们研究了这些信号，并化学合成了两条DNA双链体，每条都包含不同的起始信号。在将猿猴病毒40（SV40）小肿瘤抗原基因（SV40 t基因）克隆到pBR322质粒DNA之前，已将它们插入到距ATG起始密码子不同距离的位置。含有这两个合成核糖体结合位点（RBS）之一的质粒克隆，在距SV40 t基因不同距离处，通过免疫、电泳和蛋白水解消化分析，均产生了与天然t抗原相同的17K蛋白。这提供了一种新方法，可确保将任何连续的哺乳动物基因特异性表达克隆到细菌中，也是一种独特的体内方法，用于研究RBS与特定碱基变化的结构-功能（效率）关系。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/245f53c21edc/nar00389-0111-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/ae17189152bc/nar00389-0107-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/fc16741b8238/nar00389-0107-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/809fde91d16e/nar00389-0108-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/00d0a7e548d4/nar00389-0110-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/245f53c21edc/nar00389-0111-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/ae17189152bc/nar00389-0107-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/fc16741b8238/nar00389-0107-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/809fde91d16e/nar00389-0108-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/00d0a7e548d4/nar00389-0110-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dabf/326921/245f53c21edc/nar00389-0111-a.jpg

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本文引用的文献

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Nucleic Acids Res. 1974 Mar;1(3):331-53. doi: 10.1093/nar/1.3.331.
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Chemical synthesis of deoxyoligonucleotides by the modified triester method.采用改良三酯法化学合成脱氧寡核苷酸。
Methods Enzymol. 1980;65(1):610-20. doi: 10.1016/s0076-6879(80)65063-0.
3
The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments.
使用原核表达载体对化学合成的人γ干扰素基因进行高水平表达。
Proc Natl Acad Sci U S A. 1984 Apr;81(8):2290-4. doi: 10.1073/pnas.81.8.2290.
4
Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter.基因表达：噬菌体T5（T5P25）早期启动子的化学合成与分子克隆
Nucleic Acids Res. 1983 Sep 10;11(17):5921-40. doi: 10.1093/nar/11.17.5921.
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Proc Natl Acad Sci U S A. 1985 Jul;82(14):4587-91. doi: 10.1073/pnas.82.14.4587.
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The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.使用硫代磷酸酯修饰的DNA快速高频产生寡核苷酸定向突变。
Nucleic Acids Res. 1985 Dec 20;13(24):8765-85. doi: 10.1093/nar/13.24.8765.
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