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

在人类中，60%的治疗药物是通过改变细胞表面蛋白质的活性来发挥作用的。这些蛋白质位于膜中--一层有序的脂肪(脂)--不出所料，它们被称为完整的膜蛋白。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开发针对关键靶点-人膜蛋白的新药。要确定蛋白质的结构，首先必须能够制造出毫克量的蛋白质。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糟糕。这在很大程度上是由于酵母和哺乳动物细胞膜脂含量的显著差异造成的。特别是，酵母不产生胆固醇--一种已被证明影响许多哺乳动物膜蛋白(包括受体、转运体和离子通道)的活性和稳定性的脂类。在这里，毕赤酵母将被基因改造，这样它就可以制造胆固醇。然后，将测试这种经过改造的菌株制造哺乳动物膜蛋白的能力。如果成功，这种胆固醇生物合成酵母将为制造毫克量的许多哺乳动物膜蛋白提供一个优化的环境，并将为直接导致基于结构的药物设计的结构研究奠定基础。

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.