Purification and characterization of gingipains.
Purification and characterization of gingipains.
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DOI：
10.1002/0471140864.ps2120s49
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发表时间：
2007-08-01
影响因子：
--
通讯作者：
Nguyen, Ky-Anh
中科院分区：
文献类型：
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作者：
Potempa, Jan;Nguyen, Ky-Anh
牙龈蛋白酶是由牙龈卟啉单胞菌大量产生的半胱氨酸蛋白酶，它们共同构成该生物体牙周病发病机制中的重要毒力因子。本发明描述了一种从牙龈卟啉单胞菌菌株HG 66的生长培养基中纯化牙龈卟啉菌蛋白酶的有效方法，沿着了用于生物体生长和纯化蛋白酶的基本表征的详细方案。纯化过程包括丙酮沉淀，然后通过凝胶过滤将高分子量牙龈卟啉菌蛋白酶（Kgp和HRgpA）与RgpB分离。在Arg-Sepharose上，通过用赖氨酸（Kgp）和精氨酸（HRgpA）洗脱剂从亲和基质中的差示洗脱，进一步分离Kgp和HRgpA。从这些程序中获得的牙龈卟啉酶是稳定的，可以在-80 ℃下储存多年而不丧失活性。
Gingipains are cysteine proteases produced in large quantities by Porphyromonas gingivalis which together constitute important virulence factors in the pathogenesis of periodontal disease by that organism. Described is this unit is an efficient procedure for the purification of gingipains from the growth medium of P. gingivalis strain HG66, along with detailed protocols for growth of the organism and basic characterization of the purified proteases. The purification procedure consists of acetone precipitation followed by gel filtration to separate high-molecular-mass gingipains (Kgp and HRgpA) from RgpB. Kgp and HRgpA are further separated on Arg-Sepharose by the virtue of differential elution from the affinity matrix with lysine (Kgp) and arginine (HRgpA) eluant. Obtained from these procedures, the gingipains are stable and can be stored at -80 degrees C for years without loss of activity.