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Recombinant expression and characterization of Epulopiscium sp. Type B glyceraldehyde‐3‐phosphate dehydrogenase
Author(s) -
Troy Gina Marie,
Piefer Andrew John
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.853.13
Subject(s) - glyceraldehyde 3 phosphate dehydrogenase , biochemistry , dehydrogenase , escherichia coli , nad+ kinase , biology , histidine , cysteine , amino acid , gene , microbiology and biotechnology , enzyme
The objective of this project is to clone, express and kinetically characterize glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) from the bacterium, Epulopiscium sp. Type B. This huge, uncultured bacterium (~250 µm) is visible to the naked eye and grows in the gut of the herbivorous surgeonfish Naso tonganus , where it may assist in the fishes' algae digestion. Epulopiscium is significant because it is larger than most animal cells yet it does not have the cellular complexity of eukaryotes. To study this protein, and gain further insight into the physiology of the bacterium, the gene encoding GAPDH was amplified using PCR and cloned into Escherichia coli for subsequent protein production. GAPDH plays an essential role in glycolysis where it converts glyceraldehyde 3‐phosphate and NAD to 1,3‐bisphosphoglycerate and NADH. This represents the first energy producing step of glycolysis. Most characterized GAPDHs are homotetramers and contain conserved cysteine and histidine residues in the active site. GAPDH from Epulopiscium was compared to the GAPDH of Clostridium botulinum , E. coli , and Homo sapiens . The 27 amino acid spacing between the active site cysteine and histidine residues was conserved in Epulopiscium GAPDH. Furthermore, it was found that there was 92% amino acid consensus among all four GAPDHs examined. This project was funded by the Hartwick College Department of Chemistry.