Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies.
Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.
Laurent
F
F
Laboratoire de pathologie végétale, INRA-INA-PG, Paris, France. franck.laurent@ibp.u-psud.fr
Kotoujansky
A
A
Bertheau
Y
Y
D000907:Antibodies, Bacterial / Q000276:immunology; D000918:Antibody Specificity; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D002846:Chromatography, Affinity; D003429:Cross Reactions; D016974:Dickeya chrysanthemi / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D004951:Esterification; D006863:Hydrogen-Ion Concentration; D010368:Pectins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D000907:Antibodies, Bacterial; D002412:Cations; D011994:Recombinant Proteins; D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase