The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of β-glucosides encodes for a carbohydrate-specific outer membrane porin
The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of β-glucosides encodes for a carbohydrate-specific outer membrane porin
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DOI：
10.1046/j.1365-2958.1999.01191.x
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发表时间：
1999-01-01
影响因子：
3.6
通讯作者：
Benz, R
中科院分区：
文献类型：
--
作者：
Andersen, C;Rak, B;Benz, R
将来自大肠杆菌染色体的隐蔽基因bglH克隆到tacOP驱动的表达载体中。将得到的质粒转入孔蛋白缺陷型E. coli KS 26，IPTG诱导表达。BglH蛋白定位于外膜。使用标准方法将其纯化至均一。用脂质双层膜的重构实验将BglH定义为通道形成组分，即它是外膜孔蛋白。BglH的单通道电导（560 pS，在1 M KCl中）仅为E.碳水化合物在水相中的存在导致通过通道的离子转运的剂量依赖性阻断，类似于在大肠杆菌和鼠伤寒沙门氏菌的LamB（麦芽糖孔蛋白）中发现的情况，这意味着BglH是对碳水化合物摄取特异性的孔蛋白。各种不同的碳水化合物的结合常数计算从滴定实验的BglH诱导的膜电导。观察到与芳香族β-D-葡糖苷熊果苷和水杨苷以及龙胆二糖和纤维二糖的最紧密结合。麦芽寡糖与BglH的结合与它们与LamB的结合相反，因为它弱得多，表明BglH对碳水化合物的结合位点不同于LamB（麦芽孔蛋白）的结合位点。使用碳水化合物诱导的电流噪音研究了纤维五糖与BglH结合的动力学，并与纤维五糖与LamB（麦芽孔蛋白）和ScrY（蔗糖孔蛋白）结合的动力学进行了比较。
The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP-driven expression vector. The resulting plasmid was transferred into the porin-deficient E. coli strain KS26 and the protein was expressed by addition of IPTG. The BglH protein was localized in the outer membrane. It was purified to homogeneity using standard methods, Reconstitution experiments with lipid bilayer membranes defined BglH as a channel-forming component, i.e. it is an outer membrane porin. The single-channel conductance of BglH (560 pS in 1 M KCl) was only one-third of that of the general diffusion porins of E. coli outer membrane, The presence of carbohydrates in the aqueous phase led to a dose-dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E, coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates. The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH-induced membrane conductance. The tightest binding was observed with the aromatic beta-D-glucosides arbutin and salicin, and with gentibiose and cellobiose. Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin). The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate-induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).