César Sánchez J, Padrón G, Santana H, Herrera L
Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
J Biotechnol. 1998 Aug 27;63(3):179-86. doi: 10.1016/s0168-1656(98)00073-x.
When human interferon-alpha 2b (HuIFN alpha 2b) was expressed intracellularly in Escherichia coli as insoluble aggregates, a HuIFN alpha 2b molecular species of high molecular weight was detected, even after immunoaffinity chromatography and characterized by mass spectrometry and automatic sequencing. This HuIFN alpha 2b species was synthesized by an inefficient reading of the UGA natural stop codon, stopping the translation at another UGA in frame placed 10 codons downstream of the HuIFN alpha 2b stop signal. To avoid this translational readthrough process the UGA termination codon was replaced by UAA, which is frequently used in highly expressed E. coli genes. Simultaneously, almost all the HuIFN alpha 2b gene 3' noncoding region was removed. Analysis by SDS-PAGE and enzyme-linked immunosorbent assay revealed the elimination of the undesired HuIFN alpha 2b molecular species and an almost twofold increase in the expression level. These results indicate that both factors, the stop codon used and the length of the transcription unit should be taken into account when the expression in E. coli of heterologous proteins is desired.
当人α-2b干扰素（HuIFNα2b）在大肠杆菌中以不溶性聚集体形式在细胞内表达时，即使经过免疫亲和层析后，仍检测到一种高分子量的HuIFNα2b分子种类，并通过质谱和自动测序进行了表征。这种HuIFNα2b种类是由于对UGA天然终止密码子的低效读取而合成的，翻译在HuIFNα2b终止信号下游10个密码子处的另一个框内UGA处停止。为避免这种翻译通读过程，将UGA终止密码子替换为UAA，UAA常用于高表达的大肠杆菌基因中。同时，几乎去除了所有HuIFNα2b基因的3'非编码区。通过SDS-PAGE和酶联免疫吸附测定分析表明，不需要的HuIFNα2b分子种类被消除，表达水平几乎提高了两倍。这些结果表明，当期望在大肠杆菌中表达异源蛋白质时，所使用的终止密码子和转录单位的长度这两个因素都应予以考虑。
