Han Q, Lenz M, Tan Y, Xu M, Sun X, Tan X, Tan X, Tang L, Miljkovic D, Hoffman R M
AntiCancer, Inc., 7917 Ostrow Street, San Diego, California, 92111, USA.
Protein Expr Purif. 1998 Nov;14(2):267-74. doi: 10.1006/prep.1998.0955.
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.
利用聚合酶链式反应（PCR）方法从厌氧原生动物寄生虫阴道毛滴虫的基因组DNA中克隆出了高半胱氨酸α,γ-裂合酶，并将其在含有T7启动子的载体中于大肠杆菌中表达。重组高半胱氨酸α,γ-裂合酶（rHCYase）在宿主大肠杆菌细胞中作为主要蛋白质表达。通过在50℃进行热处理、用聚乙烯亚胺、聚乙二醇8000和高氯化钠进行沉淀步骤、DEAE-琼脂糖FF柱层析以及苯基-琼脂糖6 FF柱层析，将该酶纯化至约90%的纯度。最终产量大于50%，实现了约18倍的纯化。该酶是一种同四聚体，单体分子量为43K，含有磷酸吡哆醛。与恶臭假单胞菌的α,γ-裂合酶相比，阴道毛滴虫的rHCYase对高半胱氨酸具有选择性，而对半胱氨酸酶活性极低，恶臭假单胞菌的α,γ-裂合酶对高半胱氨酸具有非常高的半胱氨酸酶活性。阴道毛滴虫和恶臭假单胞菌的α,γ-裂合酶在苯基-琼脂糖6 FF柱上很容易分离，阴道毛滴虫的酶先洗脱。rHCYase在高达50℃时稳定，在pH值6 - 8的范围内有活性。这些在大肠杆菌中高重组表达、简单有效的高产纯化程序以及对高半胱氨酸相对于半胱氨酸的高相对特异性等特性，使得rHCYase成为用于诊断高同型半胱氨酸血症的有前途的候选物，高同型半胱氨酸血症已被证明是所有类型心血管疾病发病和死亡的主要危险因素。
