Expression of ABC Transporters in Pichia pastoris(RMI)

描述（由申请人提供）：atp结合盒（ABC）膜转运蛋白家族在人类中有超过40个成员。这些蛋白在不同的器官和细胞类型中运输许多重要的生理底物，如脂质、肽、离子和异种生物，从而发挥关键的生理作用。ABC转运蛋白的重要作用被以下事实所强调：这些转运蛋白中的一些失活突变会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的过度表达会导致细胞对药物和外源药物产生耐药性，包括用于多种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，以及相关人类病理缺陷的性质。此外，关于这些哺乳动物转运蛋白的结构信息非常少。我们之前已经成功地在酵母毕氏酵母中表达了大量具有生物活性的ABCB和ABCC家族成员，并且已经能够分离出大量纯的、具有生物活性的ABCB蛋白，其数量足以通过x射线晶体学进行结构研究。我们建议扩展这些研究，系统地表达所有与人类疾病相关的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，以及纯化大量用于生化和结构研究的单个蛋白质。重要的是，我们提出了一种增量方法，所有有用的试剂都将在我们的实验室可用并验证后提供给科学界。纯化和重组这些转运体的实验负担将由其他对特定转运体有特殊兴趣和以前经验的实验室分担。

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).