Gamer Martin, Fröde David, Biedendieck Rebekka, Stammen Simon, Jahn Dieter
Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Appl Microbiol Biotechnol. 2009 Apr;82(6):1195-203. doi: 10.1007/s00253-009-1952-5. Epub 2009 Mar 24.
Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.
基于噬菌体T7 RNA聚合酶的基因表达系统通常是高水平生产重组蛋白的最终选择。在过去十年中，革兰氏阳性菌巨大芽孢杆菌被确立为用于细胞内和细胞外生产异源蛋白的有用宿主。在本文中，我们报道了一种用于巨大芽孢杆菌的依赖T7 RNA聚合酶的表达系统的开发。该系统用来自维多利亚水母的绿色荧光蛋白（GFP）作为细胞内模型蛋白以及罗伊氏乳杆菌蔗糖酶作为细胞外模型蛋白，对胞质和分泌蛋白的生产进行了评估。在诱导T7 RNA聚合酶基因表达后，GFP在摇瓶培养中迅速在细胞内高水平积累，最高可达50 mg/l。添加利福平以抑制巨大芽孢杆菌RNA聚合酶导致GFP稳定性增加。罗伊氏乳杆菌蔗糖酶也成功产生并分泌（最高可达20 U/l）到培养上清液中。然而，该蛋白在细胞内的平行积累表明与Sec依赖性蛋白转运过程相关的局限性。
