通过固定化弹性蛋白柱层析法纯化人白细胞弹性蛋白酶和组织蛋白酶G。

Purification of human leukocyte elastase and cathepsin G by chromatography on immobilized elastin.

作者信息

Viscarello B R, Stein R L, Kusner E J, Holsclaw D, Krell R D

出版信息

Prep Biochem. 1983;13(1):57-67. doi: 10.1080/00327488308068735.

Abstract

Human leukocyte elastase and cathepsin G were isolated from purulent sputum by a simple procedure involving chromatography on elastin-agarose. Salt extracts of sputum were prepared, treated with DNase, and the precipitate which formed extracted and applied to a column of soluble elastin-Sepharose 4B. Contaminating protein was eluted with 50 mM Tris, 50 mM NaCl, pH 8.0 and then two column volumes of 50 mM acetate, 1.0 M NaCl, pH 5.0. The tightly bound elastase and cathepsin G together with a trypsin-like serine protease could finally be eluted with 50 mM acetate, 1.0 M NaCl, 20% DMSO, pH 5.0. Resolution of the proteases was accomplished by cation-exchange chromatography. Disc gel electrophoresis established the purity of elastase and cathepsin G and confirmed the existence of several isozymes for each.

摘要

通过一项简单的程序，即利用弹性蛋白琼脂糖进行层析，从脓性痰中分离出了人白细胞弹性蛋白酶和组织蛋白酶G。制备痰液的盐提取物，用DNA酶处理，形成的沉淀物经提取后应用于可溶性弹性蛋白-琼脂糖4B柱。用50 mM Tris、50 mM NaCl、pH 8.0洗脱污染蛋白，然后用两柱体积的50 mM乙酸盐、1.0 M NaCl、pH 5.0洗脱。最终，紧密结合的弹性蛋白酶、组织蛋白酶G以及一种胰蛋白酶样丝氨酸蛋白酶可用50 mM乙酸盐、1.0 M NaCl、20%二甲基亚砜、pH 5.0洗脱。通过阳离子交换层析实现蛋白酶的分离。圆盘凝胶电泳确定了弹性蛋白酶和组织蛋白酶G的纯度，并证实了每种酶都存在几种同工酶。