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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Han Q,Lenz M,Tan Y,Xu M,Sun X,Tan X,Tan X,Tang L,Miljkovic D,Hoffman RMdoi
10.1006/prep.1998.0955subject
Has Abstractpub_date
1998-11-01 00:00:00pages
267-74issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(98)90955-Xjournal_volume
14pub_type
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