Mietzner T A, Bolan G, Schoolnik G K, Morse S A
J Exp Med. 1987 Apr 1;165(4):1041-57. doi: 10.1084/jem.165.4.1041.
This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.
本报告描述了一种纯化淋病奈瑟菌和脑膜炎奈瑟菌表达的主要铁调节蛋白（MIRP）的方法。该纯化过程包括通过在铁限制培养基上培养生物体来使MIRP最大程度表达；通过超声处理进行细胞破碎，随后进行离心分级分离；用阳离子去污剂十六烷基三甲基溴化铵选择性溶解MIRP；在这种去污剂存在下进行阳离子交换色谱；以及凝胶过滤色谱。通过该技术纯化的MIRP在SDS-PAGE分析时迁移为单一条带。纯化的MIRP显示出异常碱性的等电点，该值大于9.35。进一步的生化分析揭示了从奈瑟菌属的两种致病物种中分离出的这种蛋白质具有高度保守性。例如，脑膜炎奈瑟菌和淋病奈瑟菌MIRP的氨基酸组成几乎相同，氨基末端序列分析表明两者在第48个残基之前具有相同的一级序列。令人惊讶的是，MIRP的前五个NH2末端残基与淋病奈瑟菌孔蛋白I的前五个残基具有同源性。纯化的MIRP制剂呈现出特征性的粉红色，让人联想到碱性铁结合蛋白乳铁蛋白。这一观察结果与铁调节特性相结合，促使我们分析纯化的MIRP中的铁含量。每1摩尔MIRP检测到约0.5摩尔铁。这项研究首次表明铁与MIRP相关，这一特性可能意味着该蛋白在奈瑟菌铁同化中起直接作用。虽然MIRP的确切功能尚不清楚，但以纯净且与生物学相关的量获得这种蛋白质将有助于进一步研究阐明其病理生物学功能。
