HIGH EXPRESSION, PURIFICATION, AND PROPERTIES OF RECOMBINANT HOMOCYSTEINE ALPHA,GAMMA-LYASE

Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite T richomonas vaginalis has been cloned from genomic DNA using PCR method s and expressed in Escherichia coli with a vector containing the T7 pr omoter. The recombinant homocysteine alpha,gamma-lyase (rHCYase) is ex pressed as the major protein in the host E. coli cells. The enzyme was purified to approximately 90% purity using heat treatment at 50 degre es C, precipitation steps with polyethyleneimine, polyethylene glycol 8000, and high sodium chloride, DEAE-Sepharose FF chromatography, and phenyl-Sepharose 6 FF chromatography. The final yield was greater than 50%, which encompassed an approximate 18-fold purification. The enzym e is a homotetramer with a monomer molecular weight of 43K and contain s pyridoxal phosphate. The Trichomonas rHCYase is selective for homocy steine with respect to very low cysteinase activity in contrast to the alpha,gamma-lyase from Pseudomonas putida, which has very high cystei nase activity with respect to homocysteine. The T. vaginalis and P. pu tida alpha,gamma-lyases readily separate on a phenyl-Sepharose 6 FF co lumn with the T. vaginalis enzyme eluting first. rHCYase is stable up to 50 degrees C and active over a pH range of 6-8. These properties of high recombinant expression in E. coli, a simple and effective high-y ield purification procedure and high relative specificity for homocyst eine with respect to cysteine, make rHCYase a promising candidate to u se for the diagnosis of hyperhomocystenemia, which has been demonstrat ed to be a major risk factor for the onset and mortality of cardiovasc ular disease of all types. (C) 1998 Academic Press.