Skerra A
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Gene. 1994 Dec 30;151(1-2):131-5. doi: 10.1016/0378-1119(94)90643-2.
A generic vector, pASK75, was developed for the synthesis of foreign proteins in Escherichia coli under transcriptional control of the tetA promoter/operator. Tight regulation was achieved by placing the structural gene for the tet repressor, as a transcriptional fusion, downstream from the beta-lactamase-encoding gene (bla) on the same plasmid. Strong expression of the foreign gene was conveniently induced by adding anhydrotetracycline at a low concentration. Using the production of a recombinant murine immunoglobulin F(ab) fragment as an example, the system was shown to function independently of the host-strain background and to be extremely well repressed in the absence of the inducer. Thus, it represents an economic and independent alternative to IPTG-inducible promoter constructs. Additional features of pASK75 include a signal sequence and a multiple cloning site followed by a region encoding the Strep tag affinity peptide to facilitate purification of a bacterially produced protein.
构建了一种通用载体pASK75，用于在tetA启动子/操纵子的转录控制下在大肠杆菌中合成外源蛋白。通过将四环素阻遏物的结构基因作为转录融合体，置于同一质粒上编码β-内酰胺酶的基因（bla）下游，实现了严格调控。通过添加低浓度的脱水四环素可方便地诱导外源基因的强表达。以重组鼠免疫球蛋白F(ab)片段的生产为例，该系统显示其功能独立于宿主菌株背景，且在无诱导剂时受到极强的抑制。因此，它是IPTG诱导型启动子构建体的一种经济且独立的替代方案。pASK75的其他特性包括一个信号序列、一个多克隆位点，其后是一个编码链霉亲和肽的区域，以促进细菌产生的蛋白的纯化。
