pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3
pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3
复制标题
DOI：
10.1128/aem.00906-15
复制
发表时间：
2015-06
影响因子：
4.4
通讯作者：
Xin-Wei Yang;Huahua Jian;Feng-Ping Wang
中科院分区：
文献类型：
--
作者：
Xin-Wei Yang;Huahua Jian;Feng-Ping Wang
以深海细菌Shewanella piezotolerans WP 3的丝状噬菌体（SW 1）为载体，构建了低温诱导蛋白表达载体（pSW 2）。该载体在大肠杆菌和希瓦氏菌中稳定复制，低温下拷贝数增加。pSW 2载体可作为WP 3的互补质粒，也可用于生产具有多个血红素基团的复合细胞色素，具有应用于金属离子回收或生物修复的潜力。将WP 3中的低温诱导基因的启动子融合到该载体中，构建了一系列低温下增强蛋白表达的载体。当使用hfq基因的启动子时，获得最大的绿色荧光蛋白强度。WP 3/pSW 2系统可以从大肠杆菌中易于形成包涵体的宏基因组文库中高效地产生patatin样蛋白（PLP）。杆菌在4°C和20°C下，可溶性部分中PLP的产量分别为8.3毫克/升和4.7毫克/升培养物。此外，pSW 2载体可广泛用于其他希瓦氏菌属物种，如S. oneidensis和S.嗜冷菌
ABSTRACT A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila.