Enhanced membrane protein expression by engineering increased intracellular membrane production.
Enhanced membrane protein expression by engineering increased intracellular membrane production.
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DOI：
10.1186/1475-2859-12-122
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发表时间：
2013-12-09
影响因子：
6.4
通讯作者：
Callewaert N
中科院分区：
文献类型：
--
作者：
Guerfal M;Claes K;Knittelfelder O;De Rycke R;Kohlwein SD;Callewaert N
膜蛋白的研究经常受到这些蛋白在细胞中天然丰度低的阻碍，并且通常依赖于重组基因表达。正在使用不同的表达系统，如哺乳动物细胞、昆虫细胞、细菌和酵母，但很少有研究工作针对用于增强膜蛋白表达的特定宿主细胞定制。在这里，我们表明，通过干扰脂质合成的关键酶促步骤来增加细胞内膜的生产，从而实现酵母中膜蛋白的表达增强。我们通过删除导致内质网（ER）膜大量增殖的磷脂酸磷酸酶PAH 1来工程化嗜油酵母解脂耶氏酵母。对于所有八个测试的代表不同的完整的膜蛋白家族，我们获得了增强的蛋白质积累水平，并在某些情况下，增强蛋白水解完整性的pepah 1菌株。我们更详细地分析了腺苷A2 AR G-蛋白偶联受体的情况，发现伴随诱导的未折叠的蛋白质反应在pepah 1菌株增强了受体的特异性配体结合活性。这些数据表明，一个改进的质量控制机制，膜蛋白积累在酵母细胞增殖的ER。我们的结论是，重定向脂肪酸的代谢通量远离三酰甘油和硬脂酸酯的存储对膜磷脂合成的PAH 1基因失活，提供了一个有价值的方法来提高真核细胞膜蛋白的生产。作为对膜蛋白量的这种改善的补充，UPR共诱导进一步提高了膜蛋白在其适当折叠和生物活性方面的质量。重要的是，由于这些途径在所有真核生物中都是保守的，因此在其他生物技术感兴趣的细胞类型（如昆虫细胞和哺乳动物细胞）中研究类似的工程方法将是有意义的。
Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.
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影响因子：
14.9
作者：
Hartner FS;Ruth C;Langenegger D;Johnson SN;Hyka P;Lin-Cereghino GP;Lin-Cereghino J;Kovar K;Cregg JM;Glieder A
通讯作者：
Glieder A
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
5.5
作者：
BAHARAEEN, S;VISHNIAC, HS
通讯作者：
VISHNIAC, HS
影响因子：
5
作者：
Blazeck, John;Reed, Ben;Alper, Hal S.
通讯作者：
Alper, Hal S.
影响因子：
4.8
作者：
Fakas, Stylianos;Qiu, Yixuan;Carman, George M.
通讯作者：
Carman, George M.