P. Lutwyche et al., AQUEOUS 2-PHASE POLYMER SYSTEMS AS TOOLS FOR THE STUDY OF A RECOMBINANT SURFACE-EXPRESSED ESCHERICHIA-COLI HEMAGGLUTININ, Applied and environmental microbiology, 61(9), 1995, pp. 3251-3255
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.