重组人中性粒细胞激活肽2（rhNAP-2）的纯化、结晶及初步X射线衍射分析

Purification, crystallization and preliminary X-ray diffraction analysis of recombinant human neutrophil-activating peptide 2 (rhNAP-2).

作者信息

Kungl A J, Machius M, Huber R, Schwer C, Lam C, Aschauer H, Ehn G, Lindley I J, Auer M

机构信息

Sandoz Research Institute, Wien, Austria.

出版信息

FEBS Lett. 1994 Jun 27;347(2-3):300-3. doi: 10.1016/0014-5793(94)00573-7.

DOI:10.1016/0014-5793(94)00573-7

PMID:8034022

Abstract

The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 A, b = 39.5 A and c = 95.3 A. A packing density of 3.8 A3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 A on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.

摘要

人中性粒细胞的强效激活剂和趋化因子——中性粒细胞激活肽2（NAP - 2），已在大肠杆菌中克隆并表达。该蛋白已通过包括反相高效液相色谱在内的一系列色谱技术纯化至同质状态（> 98%）。重组人NAP - 2（rhNAP - 2）的生物学活性通过诱导人中性粒细胞释放弹性蛋白酶来表征，结果发现其与天然NAP - 2相当。rhNAP - 2已通过悬滴气相扩散法结晶。晶体属于空间群P222，晶胞参数为a = 30.8 Å，b = 39.5 Å，c = 95.3 Å。假设每个不对称单元有一个分子时，堆积密度为3.8 Å³/Da，溶剂含量约为68%。这些晶体在传统X射线源上显示出能衍射到2.0 Å以上。它们对X射线稳定数天，因此适合进行高分辨率结构测定。