Shatzman A R, Gross M S, Rosenberg M
SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, USA.
Curr Protoc Mol Biol. 2001 May;Chapter 16:Unit16.3. doi: 10.1002/0471142727.mb1603s11.
In the expression system described here, plasmids (pSKF) utilize regulatory signals--such as the powerful promoter pL--from the bacteriophage lambda. Transcription from pL can be fully repressed and plasmids containing it are thus stabilized by the lambda repressor, cI. The repressor is supplied by an E. coli host which contains a integrated copy of a portion of the lambda genome. This so-called defective lysogen supplies the lambda regulatory proteins cI and N but does not provide the lytic components that would normally lead to cell lysis. Thus, cells carrying these plasmids can be grown initially to high density without expression of the cloned gene and subsequently induced to synthesize the product upon inactivation of the repressor. This system also ensures that pL-directed transcription efficiently traverses any gene insert, which is accomplished by providing the phage lambda antitermination function, N, to the cell and by including on the pL transcription unit a site necessary for N utilization (Nut site). The N protein interacts with and modifies the RNA polymerase at the Nut site so as to block transcription termination at distal sites in the transcription unit. In order to express the coding sequence, efficient ribosome-recognition and translation-initiation sites have been engineered into the pL transcription unit. Expression occurs after temperature or chemical induction inactivates the repressor (see first and second basic protocols). Restriction endonuclease sites for insertion of the desired gene have been introduced both upstream and downstream from an ATG initiation codon. Thus, the system allows either direct expression or indirect expression (via protein fusion) of any coding sequence, thereby potentially allowing expression of any gene insert. Protocols describe direct expression of "authentic" gene products, as well as heterologous genes fused to highly expressed gene partners generates chimeric proteins that differ from the native form. In the latter case, the fusion partner can be removed to obtain an unfused version of the gene product.
在此处描述的表达系统中，质粒（pSKF）利用来自噬菌体λ的调控信号，如强大的启动子pL。来自pL的转录可被完全抑制，因此含有它的质粒通过λ阻遏蛋白cI得以稳定。阻遏蛋白由含有λ基因组一部分整合拷贝的大肠杆菌宿主提供。这种所谓的缺陷溶原菌提供λ调控蛋白cI和N，但不提供通常会导致细胞裂解的裂解成分。因此，携带这些质粒的细胞最初可以高密度生长而不表达克隆基因，随后在阻遏蛋白失活时被诱导合成产物。该系统还确保pL指导的转录能有效地穿过任何基因插入片段，这是通过向细胞提供噬菌体λ抗终止功能N并在pL转录单元中包含N利用所需的位点（Nut位点）来实现的。N蛋白在Nut位点与RNA聚合酶相互作用并对其进行修饰，从而阻止转录单元远端位点的转录终止。为了表达编码序列，已在pL转录单元中设计了高效的核糖体识别和翻译起始位点。在温度或化学诱导使阻遏蛋白失活后发生表达（见第一个和第二个基本方案）。用于插入所需基因的限制性内切酶位点已被引入到ATG起始密码子的上游和下游。因此，该系统允许任何编码序列的直接表达或间接表达（通过蛋白质融合），从而有可能表达任何基因插入片段。方案描述了“真实”基因产物的直接表达，以及与高表达基因伙伴融合的异源基因产生与天然形式不同的嵌合蛋白。在后一种情况下，可以去除融合伙伴以获得基因产物的未融合版本。
