Expression of ABC Transporters in Pichia pastoris

描述（由申请人提供）：膜转运蛋白的ATP结合盒（ABC）家族在人体内有40多个成员。这些蛋白质履行关键的生理作用，因为它们在不同的器官和细胞类型中转运许多重要的生理底物，如脂质、肽、离子和外源性物质。ABC转运蛋白的重要作用突出表现在以下事实：这些转运蛋白中的几种失活突变导致人类衰弱性疾病，包括囊性纤维化、免疫缺陷、失明以及脂质和胆固醇代谢缺陷。此外，ABC转运蛋白如P-糖蛋白和MRP的过表达导致细胞对药物和外源性物质的抗性，包括用于许多类型的人类癌症化疗的药物。ABC转运蛋白功能的许多方面仍然知之甚少，包括生理底物的身份，运输机制，以及相关人类病理学缺陷的性质。此外，这些哺乳动物转运蛋白的结构信息很少。我们以前已经成功地表达了大量的生物活性成员的ABCB和ABCC家族的酵母毕赤酵母，并已能够分离出大量的纯的，生物活性的ABCB蛋白质的数量足以通过X射线晶体学的结构研究。我们建议扩展这些研究，并在巴斯德毕赤酵母中系统地表达与人类疾病相关的所有ABC转运蛋白。该提案的具体目标包括：鉴定表达大量蛋白质的克隆，表征这些蛋白质的功能状态，以及纯化大量单个蛋白质用于生物化学和结构研究。重要的是，我们提出了一种渐进的方法，所有有用的试剂都将在我们实验室得到验证后提供给科学界。纯化和重组这些转运蛋白的实验负担将由其他实验室分担，这些实验室对特定的转运蛋白有特殊的兴趣和先前的经验。

DESCRIPTION (provided by applicant): The ATP-binding cassette (ABC) family of membrane transporters has over 40 members in humans. These proteins fulfill key physiological roles as they transport a number of important physiological substrates such as lipids, peptides, ions, and xenobiotics in different organs and cell types. The important role of ABC transporters is highlighted by the fact that inactivating mutations in several of these transporters cause debilitating diseases in humans, including cystic fibrosis, immunodeficiency, blindness, and defects in lipid and cholesterol metabolism. Also, overexpression of ABC transporters such as P-glycoproteins and MRP cause cellular resistance to drugs and xenobiotics, including drugs used for chemotherapy of many types of human cancers. Many aspects of ABC transporter function remain poorly understood, including the identity of physiological substrates, the mechanism of transport, and the nature of the defect in associated human pathologies. In addition, there is very little structural information available for these mammalian transporters. We have previously succeeded in expressing large amounts of biologically active members of the ABCB and ABCC family in the yeast Pichia pastoris and have been able to isolate large amounts of pure, biologically active ABCB proteins in quantities sufficient for structural studies by X-ray crystallography. We propose to extend these studies and systematically express all ABC transporters of relevance for human disease in P. pastoris. The specific aims of this proposal include: identifying clones expressing high amounts of the proteins, characterizing the functional status of these proteins, and purifying large amounts of individual proteins for biochemical and structural studies. Importantly, we propose an incremental approach where all useful reagents will be made available to the scientific community as they become available and are validated in our lab. The experimental burden of purifying and reconstituting these transporters would be shared by other labs with a special interest and previous experience with a specific transporter(s).