Swapping of Phasin Modules To Optimize the In Vivo Immobilization of Proteins to Medium-Chain-Length Polyhydroxyalkanoate Granules in Pseudomonas putida
Swapping of Phasin Modules To Optimize the In Vivo Immobilization of Proteins to Medium-Chain-Length Polyhydroxyalkanoate Granules in Pseudomonas putida
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DOI：
10.1021/bm4008937
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发表时间：
2013-09-01
影响因子：
6.2
通讯作者：
Auxiliadora Prieto, M.
中科院分区：
文献类型：
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作者：
Dinjaski, Nina;Auxiliadora Prieto, M.
PhaF是恶臭假单胞菌KT 2442的一种双模块蛋白，在聚羟基链烷酸酯（PHA）装置内表现出多种功能。其表现为相蛋白或PHA颗粒结合蛋白（通过BioF结构域），也作为参与细胞分裂过程中颗粒定位和分离的类核相关蛋白（通过C-末端结构域）。这项工作解决了PHA颗粒形成机制中的PhaI相蛋白的功能。对产生与GFP蛋白融合的重组相蛋白结构域的恶臭假单胞菌相蛋白突变细胞的落射荧光显微镜和流式细胞术研究表明，只有当低剂量的PhaF和BioF结构域或PhaI一起表达时，细胞分裂后颗粒分布才平衡，揭示了相蛋白模块的可交换性。这些发现显示了相蛋白模块生产的精确组合，其导致最佳PHA生产和颗粒定位和分布，伴随着重组蛋白在体内固定到PHA（22 mg蛋白/g PHA）。
PhaF is a bimodular protein of Pseudomonas putida KT2442 exhibiting multiple functions within the polyhydroxyalkanoate (PHA) apparatus. It behaves as phasin or PHA granule binding protein (by BioF domain) and also as nucleoid-associated protein involved in granule localization and segregation during cell division (by C-terminal domain). This work addresses the function of the PhaI phasin in the PHA granule formation machinery. Epifluorescence microscopy and flow cytometry studies of P. putida phasin mutant cells producing recombinant phasin domains fused to GFP protein demonstrated a balanced granule distribution after cell division only when low dosage of PhaF, and BioF domain or PhaI, are expressed together, revealing the exchangeability of phasins modules. These findings show the precise combination of phasin module production that leads to the optimal PHA production and granule localization and distribution, concomitantly to in vivo immobilization of recombinant proteins to PHA (22 mg of protein/g PHA).