Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability
Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability
复制标题
DOI：
10.1016/s0378-1119(98)00020-1
复制
发表时间：
1998-03-16
期刊：
影响因子：
3.5
通讯作者：
Osburne, MS
中科院分区：
文献类型：
--
作者：
Grossman, TH;Kawasaki, ES;Osburne, MS
E.已知利用Inc操纵子控制元件来调节基因表达的大肠杆菌重组表达系统产生一定量的未诱导的（泄漏的）基因表达。以前，我们表明，高水平的未诱导的基因表达是pET表达系统不稳定的主要原因。在这里，我们表明，pET系统，其中噬菌体T7 RNA聚合酶基因是通过公司操纵子控制元件表达，表现出泄漏的表达，显着增加，在复杂的培养基中生长的细胞进入稳定期。此外，我们发现这种现象也发生在染色体lac操纵子上。进一步的研究表明，稳定期泄漏的表达需要环AMP，并在对数期细胞中加入环AMP和醋酸盐在pH 6.0的大量泄漏的表达可以受到影响。最后，其他同基因cya和野生型主机的比较表明，表达稳定性和质粒的维护cya主机大大增强，即使当细胞在非选择培养基中反复传代。这些发现都提供了一种方法，以提高稳定性的Inc为基础的重组表达系统，并表明，在诱导剂的情况下，去阻遏的Inc操纵子可能是一个一般的细胞响应营养限制的一部分。(C)1998年Elsevier Science B.V.
E. coli recombinant expression systems that utilize Inc operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression. Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system. We show here that the pET system, in which the phage T7 RNA polymerase gene is expressed via Inc operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase. Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well. Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH 6.0. Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells are passaged repeatedly in non-selection medium. These findings both provide a method to enhance the stability of Inc-based recombinant expression systems, and suggest that derepression of the Inc operon in the absence of inducer may be part of a general cellular response to nutrient limitation. (C) 1998 Elsevier Science B.V.