High expression, purification, and properties of recombinant homocysteine alpha, gamma-lyase.
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.
Han
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AntiCancer, Inc., 7917 Ostrow Street, San Diego, California, 92111, USA.
Lenz
M
M
Tan
Y
Y
Xu
M
M
Sun
X
X
Tan
X
X
Tan
X
X
Tang
L
L
Miljkovic
D
D
Hoffman
R M
RM
D000818:Animals; D013437:Carbon-Sulfur Lyases / Q000737:chemistry*; D002318:Cardiovascular Diseases / Q000503:physiopathology; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006710:Homocysteine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020138:Hyperhomocysteinemia / Q000175:diagnosis; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry; D016958:Pseudomonas putida / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D014246:Trichomonas vaginalis / Q000201:enzymology*
D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006710:Homocysteine; D011732:Pyridoxal Phosphate; D008715:Methionine; D013437:Carbon-Sulfur Lyases; D003545:Cysteine