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.
Highlights
Our ability to accurately assign gene function lags far behind the tremendous progress made in DNA sequencing
The combined results at this stage provide putative functions and probable protein identities, and were treated as hypotheses, which we explored using standard recombinant DNA technology and traditional in vitro biochemistry: the proteins identified by mass spectrometry were cloned, expressed in Escherichia coli, purified using an epitope tag and subjected to in vitro biochemical analysis
Total protein lysate was prepared from H. pylori ATCC 26695 grown on Trypticase Soy Agar (TSA) plates, and the clarified lysate was incubated with six substrate-coated nano-particles as described in Materials and Methods
Summary
Our ability to accurately assign gene function lags far behind the tremendous progress made in DNA sequencing. Given the rapid increases in the production of DNA sequence data associated with microbiomes and meta-genomes, this issue becomes ever more acute. To address this general issue, we initiated COMBREX Edu) in order to coordinate the efforts of computational and experimental biologists, and to serve as a standard repository for protein function data (predictions, hypotheses and experimental validations) [2] As part of this effort, COMBREX has: (1) created the Gold Standard Database of experimentally characterized proteins (GSDB) in conjunction with UniProt, NCBI and JCVI [3]; (2) traced the annotated functions of 15% of microbial proteins in COMBREX to their experimental sources; (3) developed a novel gene recommendation system to encourage new experiments for those proteins that would have implications for the largest number of additional proteins; and (4) directly funded the experimental testing of numerous microbial proteins using a novel small-grant model
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