Hou Y T, Lin J X, Zhou J H, Cui H
J Interferon Res. 1986 Aug;6(4):437-43. doi: 10.1089/jir.1986.6.437.
A plasmid carrying the lambda PL promoter was constructed to express efficiently unfused human alpha D-interferon (HuIFN-alpha D) in Escherichia coli using a TGATG site in its signal sequence, which occurs also in the lambda DNA sequence. The unfused nature of HuIFN-alpha D expressed by pBV867 in E. coli (BMH 71-18) was confirmed by the following evidence: first, the purified IFN showed a single band of 19.5K in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); a 27K band, representing lambda N-HuIFN-alpha D fusion protein, was not detected. Second, the peak of IFN activity coincided with the 19.5K protein. Third, the peak of absorbent material for human leukocyte IFN antibody coincided with that of IFN activity. Finally, amino-terminal sequencing of purified IFN demonstrated an unfused HuIFN-alpha D. This suggests that E. coli is able to process the signal sequence of HuIFN-alpha D. Studies on the mechanism of "translational coupling" initiation of gene expression were carried out by the construction of two hybrid plasmids and titration of the IFN activities produced by them. The level of expression by the ATG-TGATG initiation mode was found to be six times higher than that of the single ATG mode.
构建了一个携带λPL启动子的质粒，以便利用其信号序列中的TGATG位点在大肠杆菌中高效表达未融合的人αD干扰素（HuIFN-αD），该位点也存在于λDNA序列中。通过以下证据证实了pBV867在大肠杆菌（BMH 71-18）中表达的HuIFN-αD的未融合性质：首先，纯化的干扰素在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中显示出一条19.5K的单带；未检测到代表λN-HuIFN-αD融合蛋白的27K条带。其次，干扰素活性峰值与19.5K蛋白的峰值一致。第三，人白细胞干扰素抗体的吸附物质峰值与干扰素活性峰值一致。最后，纯化干扰素的氨基末端测序证明是未融合的HuIFN-αD。这表明大肠杆菌能够加工HuIFN-αD的信号序列。通过构建两种杂交质粒并滴定它们产生的干扰素活性，对基因表达的“翻译偶联”起始机制进行了研究。发现由ATG-TGATG起始模式的表达水平比单一ATG模式高六倍。
