Enrichment and purification of proteins of Haemophilus influenzae by chromatofocusing.
Haemophilus influenzae is a bacterium of pharmaceutical interest of which the entire genome has been sequenced. Identification of low-abundance proteins in a two-dimensional map is important for the detection of new drug targets. We applied chromatography on Polybuffer Exchanger (chromatofocusing) in order to fractionate and enrich H. influenzae proteins, possibly low-copy-number gene products, from larger volumes. Two proteins, major ferric iron-binding protein (HI0097) and 5'-nucleotidase (HI0206) were obtained in pure form and hypothetical protein HI0052 was purified to near homogeneity by this single purification step. Four other proteins, aspartate ammonia lyase (HI0534), peptidase D (HI0675), elongation factor Ts (HI0914) and 5-methyltetrahydropteroyltriglutamate methyltransferase (HI1702), were strongly enriched so that chromatography on Polybuffer Exchanger can be used as an initial step for their isolation. Approximately 125 proteins were identified in the fractions collected from the column. Seventy of these were for the first time identified after chromatography on Polybuffer Exchanger. The proteins enriched by the chromatofocusing step include both low-abundance as well as high-copy-number gene products. They do not belong to a single protein class and the majority of them are enzymes with various functions. The results include a list and a two-dimensional map of the proteins enriched by chromatofocusing. They may be useful in the search of drug targets and in the design of purification protocols for the isolation of homologous proteins from related microorganisms.
Fountoulakis
M
M
F. Hoffmann-La Roche Ltd., Pharma Division, Basel, Switzerland.
Langen
H
H
Gray
C
C
Takács
B
B
D001426:Bacterial Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006193:Haemophilus influenzae / Q000737:chemistry*; D007525:Isoelectric Focusing; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins