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首页> 外文期刊>Archives of Biochemistry and Biophysics >Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: High-yield expression and purification of human P-glycoprotein
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Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: High-yield expression and purification of human P-glycoprotein

机译：在啤酒酵母中使用化学伴侣来增强异源膜蛋白表达：高产量表达和纯化人P糖蛋白

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Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae, Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0.4-0.7 mu mol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated, By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone, Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp, Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K-M(MgATP),V-max, and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies. (C) 2000 Academic Press. [References: 53]
机译：利用人类P-糖蛋白（P-gp），我们研究了增强啤酒酵母中ATP结合盒转运蛋白异源表达的方法，将人类多药抗性基因MDR1 cDNA置于一个高复制2 mu酵母表达质粒的控制下诱导型GAL1启动子或强组成型PMA1启动子的表达，其中P-gp以功能形式表达。表达P-gp的酵母细胞对缬霉素具有抗性。酵母膜中P-gp的基础ATPase活性为0.4-0.7μmol/ mg / min，表明其出色的功能性。在缺乏蛋白酶的菌株BJ5457中表达的P-糖蛋白在质膜中发现，并且没有被N-糖基化。通过使用PMA1启动子，P-gp可以表达为总膜蛋白的3％。当细胞在作为化学分子伴侣的10％甘油存在下生长时，表达水平可以进一步提高至8％。类似地，甘油增强了GAL1启动子控制下表达的P-gp的蛋白质水平。已证明甘油能增强P-gp的翻译后稳定性，通过金属亲和色谱纯化带有聚组氨酸标签的P-gp，并以毫克量重构为脂质体，并对其ATPase活性进行了表征。观察到流失数高达12 s（-1）。动力学参数K-M（MgATP），V-max和药物激活取决于蛋白脂质体的脂质组成和测定的pH，与从哺乳动物来源纯化的P-gp相似。总之，我们开发了一种经济高效的功能性P-gp异源表达系统，该系统可用于生产大量正常的和突变的P-gp形式，用于结构和机理研究。 （C）2000年学术出版社。 [参考：53]

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