Szczygieł Małgorzata, Markiewicz Marcin, Szafraniec Milena, Zuziak Roxana, Urbańska Krystyna, Fiedor Leszek
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Acta Biochim Pol. 2017;64(3):437-443. doi: 10.18388/abp.2017_2299. Epub 2017 Sep 6.
All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP-binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification. The aim of this study was to optimize the methods for detection and isolation of BCRP-enriched fractions obtained from animal tissue samples. In this report we describe an optimization of isolation of a BCRP-enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools.
所有生物体都面临着众多应激因素，其中包括有害的外源性物质。这些化合物的种类繁多，因此在进化过程中，不同组织层次上发展出了多种生物防御机制。其中一种机制涉及ABC超家族中一个进化上保守的转运蛋白家族，在从细菌到人类的大多数物种中都能找到。这种转运蛋白的一个重要例子是乳腺癌耐药蛋白（BCRP/ABCG2），它是一种典型的整合膜蛋白。它在多种外源性物质的吸收、分布和消除中起关键作用，这些外源性物质包括化疗药物，并且它还与多药耐药性有关。它还能抵御饮食来源的光毒性叶绿素衍生物。BCRP是一种半转运蛋白，由一个跨膜结构域和一个ATP结合结构域组成，跨膜结构域由六个α螺旋形成一个特征性的孔结构，ATP结合结构域提供ATP水解产生的能量，这是底物主动转运所必需的。BCRP的分离仍然不是一件容易的事，因为它在水中不溶以及膜筏的存在给纯化过程带来了严重的方法学和技术挑战。本研究的目的是优化从动物组织样本中获得富含BCRP组分的检测和分离方法。在本报告中，我们描述了一种富含BCRP膜组分的分离优化方法，该方法适用于使用分子生物学工具进行进一步的蛋白质定量和定性分析。
