Aqueous two-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, 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.
Lutwyche
P
P
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Norris-Jones
R
R
Brooks
D E
DE
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003911:Dextrans; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005362:Ficoll; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014867:Water
D018829:Adhesins, Bacterial; D003911:Dextrans; D029968:Escherichia coli Proteins; C090012:HRA1 protein, E coli; D006388:Hemagglutinins; D011994:Recombinant Proteins; D014867:Water; D005362:Ficoll; D011092:Polyethylene Glycols