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Genetic manipulation of the sterol content of Pichia pastoris : a designer host for mammalian membrane protein expression

毕赤酵母甾醇含量的基因操作：哺乳动物膜蛋白表达的设计宿主

基本信息

批准号：
BB/E017061/1
负责人：
Niall Fraser
金额：
$ 13.04万
依托单位：
University of Glasgow
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE017061%2F1
关键词：
Genetic manipulation sterol content Pichia

项目摘要

In humans, 60% of all therapeutic drugs work by altering the activity of proteins found on the surface of cells. These proteins are located in a membrane - an ordered layer of fat (lipid) - and are, unsurprisingly, known as integral membrane proteins. Very few mammalian membrane proteins have had their 3-dimesional structures determined. This has largely prevented the creation of new drugs against key target, human membrane proteins by design strategies based on the structure of the protein. To determine the structure of a protein, it is first necessary to be able to make the protein in milligram amounts. Yeasts are useful host cells for making many mammalian water-soluble proteins. In contrast, they are rather poor at synthesising mammalian membrane proteins. This is in no small part due to substantial differences in the lipid content of membranes from yeast and mammalian cells. In particular, yeast do not make cholesterol - a lipid which has been shown to affect the activity and stability of many mammalian membrane proteins (including receptors, transporters and ion channels). Here, the yeast Pichia pastoris is going to be genetically manipulated so that it can make cholesterol. This modified strain will then be tested for its ability to make mammalian membrane proteins. If successful, the cholesterol biosynthetic yeast will provide an optimised environment for making milligram quantities of many mammalian membrane proteins, and will underpin structural studies that will lead directly to structure-based drug design.

在人类中，60%的治疗药物通过改变细胞表面蛋白质的活性来发挥作用。这些蛋白质位于膜中-一种有序的脂肪层（脂质）-并且，不出所料，被称为整合膜蛋白。很少有哺乳动物膜蛋白的三维结构被确定。这在很大程度上阻止了通过基于蛋白质结构的设计策略来创建针对关键靶点人类膜蛋白的新药。为了确定蛋白质的结构，首先需要能够以毫克的数量制造蛋白质。酵母菌是制造许多哺乳动物水溶性蛋白质的有用宿主细胞。相比之下，它们在合成哺乳动物膜蛋白方面相当差。这在很大程度上是由于酵母细胞和哺乳动物细胞膜的脂质含量存在很大差异。特别是，酵母不制造胆固醇-一种已被证明会影响许多哺乳动物膜蛋白（包括受体，转运蛋白和离子通道）的活性和稳定性的脂质。在这里，巴斯德毕赤酵母将被基因操纵，这样它就可以制造胆固醇。然后将测试这种修饰的菌株制造哺乳动物膜蛋白的能力。如果成功的话，胆固醇生物合成酵母将提供一个优化的环境，使毫克数量的许多哺乳动物膜蛋白，并将支持结构研究，将直接导致基于结构的药物设计。