Biedendieck Rebekka, Borgmeier Claudia, Bunk Boyke, Stammen Simon, Scherling Christian, Meinhardt Friedhelm, Wittmann Christoph, Jahn Dieter
Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, Braunschweig, Germany.
Methods Enzymol. 2011;500:165-95. doi: 10.1016/B978-0-12-385118-5.00010-4.
The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data.
革兰氏阴性菌大肠杆菌是学术界和工业界最广泛使用的重组蛋白生产宿主。革兰氏阳性菌巨大芽孢杆菌是一种越来越多地用于高产细胞内和细胞外蛋白质合成的替代宿主。在过去二十年中，已经开发了多种工具，包括基因表达质粒和生产菌株。通过原生质体转化或转接合将游离复制质粒和整合质粒引入巨大芽孢杆菌是可行的。使用His(6)和StrepII亲和标签，可以通过一步法轻松纯化细胞内或细胞外产生的蛋白质。基于木糖控制启动子P(xylA)的不同基因表达系统和各种噬菌体RNA聚合酶（T7、SP6、K1E）驱动的系统使巨大芽孢杆菌每升能够产生高达1.25克的重组蛋白。通过在生物反应器中进行高细胞密度培养，可以实现高达80g/l的生物量浓度。通过优化的基因破坏系统，可以在巨大芽孢杆菌中进行基因敲除和基因替换。为了在工业中安全应用，可以获得芽孢形成缺陷、蛋白酶缺陷以及对紫外线敏感的突变体。借助最近公布的巨大芽孢杆菌基因组序列，有可能通过基于转录组、蛋白质组、代谢组和通量组数据的系统生物学方法来表征蛋白质生产过程中的瓶颈。生物信息学平台（Megabac，http://www.megabac.tu-bs.de）整合了获得的理论和实验数据。
