Meeting Abstract
Hydrostatic pressure has a strong influence on the physiology of deep-sea animals, since many proteins do not function constantly over a pressure range of hundreds of atm. Ctenophores, or “comb jellies,” are gelatinous animals that have colonized most of the oceanic water column, from the surface to ~7 km deep, where ambient pressure is about 700 atm. We have begun to assess the functional diversity of ctenophore metabolism by cloning the glycolytic enzyme pyruvate kinase (PK) [EC 2.7.1.40] from several species living across a depth gradient. We then expressed these PK orthologs in E. coli and assayed their activity under pressure. PK was chosen based on its putatively adaptive pressure resistance in deep-sea fishes and in preliminary studies on whole ctenophore homogenates. The pressure/activity data reported here are novel with respect to invertebrates, and offer a ready comparison to fish datasets.
Overproduction of enzymes from deep-sea invertebrates for comparative physiology presents both benefits and challenges. Major advantages are (1) the option of site-directed mutagenesis, which can be used to reveal sequence features that confer pressure resistance, and (2) the ability to produce unlimited amounts of protein from a single small individual, rather than having to collect rare animals and pool substantial biomass. The most pressing challenges are (1) producing a fully native amino acid sequence, i.e. precisely as found in the animal, with no artificially appended residues, and (2) preserving the activity of heat-labile products. We fulfilled both these requirements by incorporating a highly specific protein tag cleavage system into a cold-shock expression plasmid. The resultant vector is useful for studying protein evolution in cold-adapted organisms.
