Wang Yang, Heermann Ralf, Jung Kirsten
Munich Center for Integrated Protein Science (CIPSM) at the Department of Microbiology, Ludwig-Maximilians-Universität München , 82152 Martinsried, Germany.
ACS Synth Biol. 2017 May 19;6(5):826-836. doi: 10.1021/acssynbio.6b00323. Epub 2017 Feb 10.
Natural and synthetic scaffolds support enzyme organization in complexes, and they regulate their function and activity. Here we report that CipA and CipB, two small proteins that form protein crystalline inclusions (PCIs) in the cytoplasm of Photorhabdus luminescens, can be utilized as scaffolds to efficiently incorporate exogenous proteins into PCIs. We demonstrate that Cip-tagged GFP is assembled into fluorescent PCIs in P. luminescens and that in Escherichia coli Cip scaffolds can organize GFP or/and LacZ into bioactive PCIs, which could easily be isolated for in vitro catalysis. To explore its in vivo application further, we used CipA to bring together multiple enzymes (Vio enzymes) of the violacein biosynthetic pathway. The resulting complexes were found to produce significantly higher yields of violacein and fewer byproducts than did Vio enzymes in solution. Hence, Cip scaffolds should be widely applicable to biotechnological processes both in vitro and in vivo.
天然和合成支架支持酶在复合物中的组织，并调节其功能和活性。在此，我们报告发光杆菌细胞质中形成蛋白质晶体包涵体（PCI）的两种小蛋白CipA和CipB可作为支架，有效地将外源蛋白整合到PCI中。我们证明，带有Cip标签的绿色荧光蛋白（GFP）在发光杆菌中组装成荧光PCI，并且在大肠杆菌中，Cip支架可以将GFP或/和乳糖酶（LacZ）组织成生物活性PCI，这些PCI可以很容易地分离出来用于体外催化。为了进一步探索其体内应用，我们使用CipA将紫罗碱生物合成途径的多种酶（Vio酶）聚集在一起。结果发现，与溶液中的Vio酶相比，所得复合物产生的紫罗碱产量显著更高，副产物更少。因此，Cip支架应该广泛应用于体外和体内的生物技术过程。
