Porowińska Dorota, Wujak Magdalena, Roszek Katarzyna, Komoszyński Michał
Zakład Biochemii, Wydział Biologii i Ochrony Środowiska, Uniwersytet Mikołaja Kopernika w Toruniu, Toruń.
Postepy Hig Med Dosw (Online). 2013 Mar 1;67:119-29. doi: 10.5604/17322693.1038351.
For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.
对于重组蛋白的过量生产,真核和原核表达系统都有使用。选择合适的系统除其他因素外,还取决于宿主细胞的生长速率和培养情况、靶基因的表达水平以及合成蛋白的翻译后加工。无论表达系统的类型如何,其基本元件都是载体和表达宿主。在实验室和工业规模上,用于蛋白过量生产最广泛的系统是原核系统。该系统主要基于大肠杆菌,不过越来越多地使用芽孢杆菌属。原核系统能让人在短时间内获得大量重组蛋白。简单且廉价的细菌细胞培养以及广为人知的转录和翻译机制便于这些微生物的使用。基因修饰的简易性以及许多细菌突变体的可得性是原核系统的额外优势。在本文中,我们描述了原核表达载体的结构元件。还讨论了制备靶蛋白基因的策略,这些策略可提高产量、便于重组蛋白的检测和纯化并赋予其活性。对表达系统中常作为宿主细胞使用的细菌菌株以及异源蛋白的潜在定位进行了描述。了解原核表达系统的基本元件有助于在短时间内以令人满意的量生产生物活性蛋白。