Purification and partial characterization of a transglutaminase from dog filarial parasite, dirofilaria immitis

Abstract

Transglutaminase-catalyzed reactions have been implicated to play a role in growth and development of filarial nematodes and thus could serve as target for the development of an effective chemotherapeutic agent. Characterization of this enzyme from different nematodes will be useful in better designs of enzyme-specific inhibitors. A simple three-step purification protocol was adopted to purify transglutaminase from filarial nematode Dirofilaria immitis . The three steps used were thermoprecipitation, ammonium sulfate precipitation and immunoaffinity chromatography. The antibody used for immunoaffinity chromatography was raised in rabbit against a synthetic peptide corresponding to the N-terminal sequence of a recently purified transglutaminase from Brugia malayi . About 900-fold purification was achieved in a single immunoaffinity chromatography step that yielded a 56-kDa protein as the major band in denaturing polyacrylamide gel electrophoresis. The protein eluted at this step was enzymatically active and had a specific activity of 2 U/mg protein. Several physicochemical properties such as thermostability, substrate specificity, effect of various reagents and inhibitors suggested that this enzyme is very similar to the transglutaminase purified from B. malayi , but was quite distinct from the mammalian transglutaminases. The cumulative 10,000-fold purification obtained by the current protocol suggested that the procedure could be used for the purification of transglutaminases in lower and related organisms where these vital enzymes are present in extremely low amounts (210).