Potempa Jan, Nguyen Ky-Anh
Jagiellonian University, Krakow, Poland.
Westmead Centre for Oral Health, Sydney, Australia.
Curr Protoc Protein Sci. 2007 Aug;Chapter 21:21.20.1-21.20.27. doi: 10.1002/0471140864.ps2120s49.
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.
牙龈蛋白酶是牙龈卟啉单胞菌大量产生的半胱氨酸蛋白酶，它们共同构成了该生物体在牙周病发病机制中的重要毒力因子。本单元描述了一种从牙龈卟啉单胞菌HG66菌株的生长培养基中纯化牙龈蛋白酶的有效方法，以及该生物体生长的详细方案和纯化蛋白酶的基本特性。纯化过程包括丙酮沉淀，然后进行凝胶过滤，以将高分子量牙龈蛋白酶（Kgp和HRgpA）与RgpB分离。Kgp和HRgpA通过用赖氨酸（Kgp）和精氨酸（HRgpA）洗脱剂从亲和基质中进行差异洗脱，在精氨酸琼脂糖凝胶上进一步分离。通过这些方法获得的牙龈蛋白酶很稳定，可以在-80℃下保存数年而不失活。
