Volyanik E V, Dalley A, McKay I A, Leigh I, Williams N S, Bustin S A
Surgical Unit, London Hospital Medical College, University of London, Whitechapel, England.
Anal Biochem. 1993 Oct;214(1):289-94. doi: 10.1006/abio.1993.1490.
Cell-free synthesis of preparative amounts of interleukin-6 (IL-6) in a wheat germ extract is described. Following transcription of uncapped mRNA by SP6 polymerase, protein was synthesized in a continuous flow of translation components. The system remained active for at least 50 h, producing 1.25 x 10(6) units of IL-6. Recovered IL-6 was > 80% pure, reacted with anti-IL-6 polyclonal antiserum in Western blots, and had a specific activity comparable to recombinant IL-6 purified from Escherichia coli. This is the first time biologically active IL-6 has been prepared in preparative amounts using a continuous-flow cell-free translation system and confirms the feasibility of using enhanced in vitro translation for rapid synthesis of proteins from cloned templates.
本文描述了在小麦胚芽提取物中无细胞合成制备量白细胞介素-6（IL-6）的方法。通过SP6聚合酶对无帽mRNA进行转录后，在连续流动的翻译组分中合成蛋白质。该系统至少保持活性50小时，产生1.25×10⁶单位的IL-6。回收的IL-6纯度>80%，在蛋白质免疫印迹中与抗IL-6多克隆抗血清反应，其比活性与从大肠杆菌纯化的重组IL-6相当。这是首次使用连续流动无细胞翻译系统制备出具有生物活性的制备量IL-6，并证实了使用增强的体外翻译从克隆模板快速合成蛋白质的可行性。
