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B6db references: 9790890

type Journal Article
authors Han Q, Lenz M, Tan Y, Xu M, Sun X, Tan X, Tan X, Tang L, Miljkovic D, Hoffman RM
title High expression, purification, and properties of recombinant homocysteine alpha, gamma-lyase
journal Protein Expr Purif
sel selected
ui 9790890
year (1998)
volume 14
number 2
pages 267-74
abstract Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite Trichomonas vaginalis has been cloned from genomic DNA using PCR methods and expressed in Escherichia coli with a vector containing the T7 promoter. The recombinant homocysteine alpha,gamma-lyase (rHCYase) is expressed as the major protein in the host E. coli cells. The enzyme was purified to approximately 90% purity using heat treatment at 50 degreesC, 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 enzyme is a homotetramer with a monomer molecular weight of 43K and contains pyridoxal phosphate. The Trichomonas rHCYase is selective for homocysteine with respect to very low cysteinase activity in contrast to the alpha,gamma-lyase from Pseudomonas putida, which has very high cysteinase activity with respect to homocysteine. The T. vaginalis and P. putida alpha,gamma-lyases readily separate on a phenyl-Sepharose 6 FF column with the T. vaginalis enzyme eluting first. rHCYase is stable up to 50 degreesC and active over a pH range of 6-8. These properties of high recombinant expression in E. coli, a simple and effective high-yield purification procedure and high relative specificity for homocysteine with respect to cysteine, make rHCYase a promising candidate to use for the diagnosis of hyperhomocystenemia, which has been demonstrated to be a major risk factor for the onset and mortality of cardiovascular disease of all types. Copyright 1998 Academic Press.


last changed 2008/04/10 10:39

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