Lukacsovich T, Baliko G, Orosz A, Balla E, Venetianer P
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
J Biotechnol. 1990 Mar;13(4):243-50. doi: 10.1016/0168-1656(90)90072-j.
A family of expression plasmid vectors were constructed by fusing the strong P2 promoter of the rrnB gene of Escherichia coli (coding for ribosomal RNA) to the lac operator, thereby eliminating regulatory sequences from the rrnB gene and placing the expression under lac repressor control. This promoter proved to be stronger in vivo than the well-known consensus tac promoter, and its strength could be further increased by converting the sequence to consensus. The stability of the recombinant proteins could be increased by fusion to various lengths of the N-terminal end of beta-galactosidase, or by inserting a synthetic oligonucleotide, coding for heptathreonine. A new method was developed for the stabilization of recombinant plasmids without antibiotic selection, based on the presence of an essential gene on the plasmid and its absence from the chromosome. The application of this method is illustrated by the example of a plasmid expressing human proinsulin.
通过将大肠杆菌rrnB基因（编码核糖体RNA）的强P2启动子与lac操纵子融合，构建了一系列表达质粒载体，从而去除了rrnB基因的调控序列，并使表达受lac阻遏物控制。该启动子在体内被证明比著名的共有tac启动子更强，并且通过将序列转化为共有序列，其强度可以进一步提高。通过与不同长度的β-半乳糖苷酶N末端融合，或通过插入编码七苏氨酸的合成寡核苷酸，可以提高重组蛋白的稳定性。基于质粒上存在一个必需基因而染色体上不存在该基因，开发了一种无需抗生素选择即可稳定重组质粒的新方法。以表达人胰岛素原的质粒为例说明了该方法的应用。
