AQUEOUS 2-PHASE POLYMER SYSTEMS AS TOOLS FOR THE STUDY OF A RECOMBINANT SURFACE-EXPRESSED ESCHERICHIA-COLI HEMAGGLUTININ

The surface expression of an integral membrane hemagglutinin, HRA1, cl oned 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 differ ently from both the host bacteria lacking the plasmid and the original hemagglutinating strain in several of these systems. By using molecul ar biological techniques, the origin of the partition difference was u nambiguously correlated to the expression of HRA1, providing evidence independent of the agglutination phenotype that the protein was access ible to the surrounding milieu. It Was demonstrated by using bacterial partition in charge-sensitive systems that the agglutination event wa s not likely to be due to the presence of a nonspecific positively cha rged surface protein, as HRA1-expressing clones showed no less affinit y for the relatively positive polyethylene glycol-rich upper phase tha n did control bacteria, This work demonstrates the utility of aqueous polymer two-phase systems for the study of surface-expressed recombina nt proteins, due to the sensitivity of the systems and the presence of excellent controls (the host bacteria before plasmid introduction). I n cloning and expression studies of surface-associated proteins, two-p hase aqueous polymer systems could be used as an alternative to antibo dy production for the monitoring of surface expression, and these syst ems may give valuable information on the surface exposure of the prote in.