Robben J, Massie G, Bosmans E, Wellens B, Volckaert G
Laboratory of Gene Technology, K.U. Leuven, Belgium.
Gene. 1993 Apr 15;126(1):109-13. doi: 10.1016/0378-1119(93)90597-v.
A very small plasmid vector system is described for construction and high-level production of C-terminal chloramphenicol acetyltransferase (CAT) fusion proteins in Escherichia coli. The only functional elements of the plasmid are a minimal region of the ColE1 origin of DNA replication and the Tn9 cat gene, both under control of a tac promoter. Since C-terminal fusion to CAT does not interfere with chloramphenicol (Cm) resistance, plasmids are maintained under Cm selection. Because of its small size (1392 bp), the system is especially convenient for building and expression of synthetic genes and gene fragments. This concept was utilized to generate a fusion with a synthetic gene encoding the multiple-epitope fragment from the rubella virus E1 membrane protein. Affinity-purified fusion proteins were obtained in mg amounts from 100-ml batches of culture fluid, and incorporated as a specific antigen in a rubella immunoglobulin G enzyme-linked immunosorbent assay.
本文描述了一种非常小的质粒载体系统，用于在大肠杆菌中构建和高效生产C端氯霉素乙酰转移酶（CAT）融合蛋白。该质粒的唯一功能元件是DNA复制ColE1起始位点的一个最小区域和Tn9 cat基因，二者均受tac启动子控制。由于与CAT的C端融合不影响氯霉素（Cm）抗性，质粒在Cm选择下得以维持。由于其尺寸小（1392 bp），该系统对于合成基因和基因片段的构建及表达特别方便。利用这一概念构建了与编码风疹病毒E1膜蛋白多表位片段的合成基因的融合体。从100毫升培养液批次中以毫克量获得了亲和纯化的融合蛋白，并将其作为特异性抗原用于风疹免疫球蛋白G酶联免疫吸附测定。
