title: Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori.
abstract: The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0-7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.
authors.affiliation: Dept of Biomedical Engineering, Boston University, Boston, Massachusetts, United States of America.
authors.forename: Han-Pil
authors.initials: HP
authors.lastname: Choi
authors.forename: Silvia
authors.initials: S
authors.lastname: Juarez
authors.forename: Sergio
authors.initials: S
authors.lastname: Ciordia
authors.forename: Marisol
authors.initials: M
authors.lastname: Fernandez
authors.forename: Rafael
authors.initials: R
authors.lastname: Bargiela
authors.forename: Juan P
authors.initials: JP
authors.lastname: Albar
authors.forename: Varun
authors.initials: V
authors.lastname: Mazumdar
authors.forename: Brian P
authors.initials: BP
authors.lastname: Anton
authors.forename: Simon
authors.initials: S
authors.lastname: Kasif
authors.forename: Manuel
authors.initials: M
authors.lastname: Ferrer
authors.forename: Martin
authors.initials: M
authors.lastname: Steffen
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D016480:Helicobacter pylori / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins