Mashko S V, Lebedeva M I, Podkovyrov S M, Lapidus A L, Trukhan M E
Antibiot Med Biotekhnol. 1987 Apr;32(4):248-54.
The basic results of the studies on expression of the genes of human alpha F- and beta 1-interferons in E. coli cells are presented. To synthesize the fibroblast interferon, the respective fragment of the human chromosome was cloned, the complete nucleotide sequence of the structural moiety of mature beta-interferon was determined and the genes of "hybrid (interferon-like) proteins" and "hybrid sites of ribosome binding" were constructed with control of the beta-interferon gene by the prokaryotic regulatory areas. Synthesis of beta-interferon was achieved (1.10(7)-5.10(7) IU per 1 l of the bacterial culture) with the use of the tryptophan operon promoter. A new procedure for optimization of allogenic genetic information in E. coli cells: constructing of "hybrid operons with partially overlapping genes" or artificial "overlappons" was developed following the example of the alpha F-interferon gene cloned in the Laboratory headed by E. D. Sverdlov at the Institute of Bioorganic Chemistry of the USSR Academy of Sciences. The use of this procedure enabled production of up to 5.10(7) IU/l of alpha F-interferon under the control of the lacUV5-promoter. On the basis of the newly constructed vector molecules expression of the genes of alpha F- and beta 1-interferons was amplified with the "overlappon" procedure.
本文介绍了在大肠杆菌细胞中表达人αF干扰素和β1干扰素基因的研究基本结果。为了合成成纤维细胞干扰素,克隆了人染色体的相应片段,测定了成熟β干扰素结构部分的完整核苷酸序列,并构建了“杂合(干扰素样)蛋白”和“核糖体结合杂合位点”,其β干扰素基因受原核调控区控制。利用色氨酸操纵子启动子实现了β干扰素的合成(每升细菌培养物1.10⁷ - 5.10⁷国际单位)。以苏联科学院生物有机化学研究所E.D.斯韦尔德洛夫领导的实验室克隆的αF干扰素基因为例,开发了一种优化大肠杆菌细胞中外源遗传信息的新方法:构建“部分重叠基因的杂合操纵子”或人工“重叠操纵子”。使用该方法,在lacUV5启动子控制下,αF干扰素的产量可达5.10⁷国际单位/升。基于新构建的载体分子,利用“重叠操纵子”方法扩增了αF干扰素和β1干扰素基因的表达。