Valls M, González-Duarte R, Atrian S, De Lorenzo V
Departament de Genètica, Universitat de Barcelona, Spain.
Biochimie. 1998 Oct;80(10):855-61. doi: 10.1016/s0300-9084(00)88880-x.
In view of potential biotechnological applications, eukaryotic metallothioneins (MTs) have been expressed in Escherichia coli as fusions to membrane or membrane-associated proteins such as LamB, the peptidoglycan-associated lipoprotein protein (PAL) or a hybrid Lpp/OmpA carrier sequence. The use of different anchors enables the MT moiety to be targeted into various cell compartments thus bringing the metal-binding ability of the resulting hybrids to specific sites of the cell structure. To this end, both full-size and partial sequences of the human or mouse MTs have been genetically fused to: i) the permissive site 153 of the LamB sequence, which loops out the MT to the external medium; ii) the N-terminus of a PAL variant devoid of its N-terminal cystein, which targets expression of the fusion into the periplasm; and iii) the C-terminus of Lpp-OmpA, for anchoring the MT to the outer membrane protein as an N-terminal fusion. Each type of fusion presented a distinct behavior in terms of expression, stability and ability to endow E. coli cells an enhanced accumulation of Cd2+, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions. The expression in vivo of metalloproteins bound to bacterial envelope structures opens a way to design biomass with specific metal-binding properties.
鉴于潜在的生物技术应用，真核金属硫蛋白（MTs）已在大肠杆菌中作为与膜或膜相关蛋白（如LamB、肽聚糖相关脂蛋白（PAL）或杂合Lpp/OmpA载体序列）的融合蛋白进行表达。使用不同的锚定序列可使MT部分靶向进入各种细胞区室，从而将所得杂合蛋白的金属结合能力带到细胞结构的特定部位。为此，人或小鼠MTs的全长和部分序列已被基因融合到：i）LamB序列的允许位点153，该位点使MT环出到外部介质中；ii）不含N端半胱氨酸的PAL变体的N端，其将融合蛋白的表达靶向到周质中；iii）Lpp-OmpA的C端，用于将MT作为N端融合蛋白锚定到外膜蛋白上。每种融合类型在表达、稳定性以及赋予大肠杆菌细胞增强的Cd2+积累能力方面都表现出不同的行为，这与融合蛋白的MT部分贡献的金属结合中心数量密切相关。与细菌包膜结构结合的金属蛋白在体内的表达为设计具有特定金属结合特性的生物质开辟了一条途径。
