AQUEOUS 2-PHASE POLYMER SYSTEMS AS TOOLS FOR THE STUDY OF A RECOMBINANT SURFACE-EXPRESSED ESCHERICHIA-COLI HEMAGGLUTININ
AQUEOUS 2-PHASE POLYMER SYSTEMS AS TOOLS FOR THE STUDY OF A RECOMBINANT SURFACE-EXPRESSED ESCHERICHIA-COLI HEMAGGLUTININ
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DOI：
10.1128/aem.61.9.3251-3255.1995
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发表时间：
1995-09-01
影响因子：
4.4
通讯作者：
BROOKS, DE
中科院分区：
文献类型：
--
作者：
LUTWYCHE, P;NORRISJONES, R;BROOKS, DE
从大肠杆菌O 9：H10：K99中克隆了一种完整的膜血凝素HRA 1，并在异源大肠杆菌中进行了表面表达。利用聚乙二醇-右旋糖酐和右旋糖酐-Ficoll双水相聚合物体系对大肠杆菌菌株进行了研究。在这些系统中，发现含有编码血凝素的质粒的细菌与缺乏质粒的宿主细菌和原始血凝菌株的分区不同。通过使用分子生物学技术，分区差异的起源是明确相关的HRA 1的表达，提供证据的凝集表型的蛋白质是可访问的周围环境的独立。通过在电荷敏感系统中使用细菌分区证明，凝集事件不太可能是由于非特异性带正电荷的表面蛋白的存在，因为表达HRA 1的克隆对相对阳性的富含聚乙二醇的上相显示出不低于对照细菌的亲和力，这项工作证明了水相聚合物两相系统用于研究表面表达的重组蛋白的实用性，这是由于系统的灵敏度和优良对照的存在（质粒引入之前的宿主细菌）。在表面相关蛋白的克隆和表达研究中，两相水相聚合物系统可以用作抗体生产的替代方案，用于监测表面表达，并且这些系统可以提供关于蛋白的表面暴露的有价值的信息。
The surface expression of an integral membrane hemagglutinin, HRA1, cloned from Escherichia coli O9:H10:K99 in heterologous E. coli strains was studied by utilizing a variety of polyethylene glycol-dextran and dextran-Ficoll aqueous two-phase polymer systems. Bacteria containing plasmids that encoded the hemagglutinin were found to partition differently from both the host bacteria lacking the plasmid and the original hemagglutinating strain in several of these systems. By using molecular biological techniques, the origin of the partition difference was unambiguously correlated to the expression of HRA1, providing evidence independent of the agglutination phenotype that the protein was accessible to the surrounding milieu. It Was demonstrated by using bacterial partition in charge-sensitive systems that the agglutination event was not likely to be due to the presence of a nonspecific positively charged surface protein, as HRA1-expressing clones showed no less affinity for the relatively positive polyethylene glycol-rich upper phase than did control bacteria, This work demonstrates the utility of aqueous polymer two-phase systems for the study of surface-expressed recombinant proteins, due to the sensitivity of the systems and the presence of excellent controls (the host bacteria before plasmid introduction). In cloning and expression studies of surface-associated proteins, two-phase aqueous polymer systems could be used as an alternative to antibody production for the monitoring of surface expression, and these systems may give valuable information on the surface exposure of the protein.