Purification and characterization of a recombinant Haemophilus influenzae outer membrane phosphomonoesterase e (P4).
Haemophilus influenzae is a common inhabitant of the upper respiratory tract and can cause serious infections of mucosal surfaces. Results from recent studies indicate that this pathogen possesses copious amounts of surface-localized phosphomonoesterase activity mediated by the bacterial lipoprotein e (P4). While the enzyme has previously been purified to apparent homogeneity, purification of large amounts of protein has been prevented by presence of N-terminal lipid modification. Recombinant DNA technology was employed to simultaneously replace the N-terminal lipid modification signal sequence with one for protein secretion without such modification and to place expression of the protein under the control of the T7-inducible promoter. Results from this work show that high levels of phosphomonoesterase activity were achieved after IPTG induction and purified to apparent homogeneity after two chromatography steps. Consistent with loss of the N-terminal lipid modification, the recombinant enzyme was easily extracted from the bacterial membrane and partitioned within the matrix of gel filtration chromatography resin while retaining a denatured molecular weight similar to that of wild-type e (P4). Results from physicochemical characterization suggest that the recombinant protein was similar to wild-type protein in SDS-PAGE-derived molecular weight, primary structure, substrate specificity, pH optimum, and sensitivity or resistance to various inhibitors. Acquisition of sufficient amounts of recombinant P4 was a prelude for studies to elucidate the structure and function of this unusual phosphomonoesterase.
Reilly
T J
TJ
Department of Molecular Microbiology and Immunology, University of Missouri Medical School, Columbia, Missouri 65212, USA.
Smith
A L
AL
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D004950:Esterases; D006193:Haemophilus influenzae / Q000378:metabolism*; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; C098614:e(P4) lipoprotein, Haemophilus influenzae; D004950:Esterases; D010744:Phosphoric Monoester Hydrolases