Engineering highly efficient promoters for eukaryotic protein production, tissue specific expression, and biomedical pathway analysis by Oxford Genetics Ltd

生产用于医学研究和保健的重组蛋白是生物技术的一个迅速发展的领域。然而，这些蛋白质中的许多必须由廉价的哺乳动物系统制造，以确保正确的蛋白质折叠，其产量通常比细菌系统低(通常低100-1000倍)。这在一定程度上是由于哺乳动物启动子(控制蛋白质生产的)与细菌启动子相比强度较低。细菌启动子的强度很容易预测，产生的蛋白质量往往与所用启动子与最佳共识启动子的相似程度成正比。在哺乳动物的启动子中，同样的预测无法做出，因为启动子的结构要复杂得多，因此很难知道启动子的哪些部分能产生更强的蛋白质。因此，大多数哺乳动物的表达系统都是基于自然产生的野生型启动子，这些启动子实际上是为了生理功能而进化的，而不是商业蛋白质制造。我们的初步数据显示，使用我们的技术可以开发出显著更强的启动子，这对蛋白质制造具有重要意义。在牛津遗传有限公司，我们开发了一种名为普罗米修斯的生物选择克隆系统，以筛选大量(&gt；4000)重组哺乳动物启动子。这项技术允许对多种强大的“野生型”哺乳动物启动子进行“洗牌”和筛选，以确定比目前的黄金标准CMV和CAG产生更多蛋白质的新的复合启动子。初步结果令人兴奋，在这个项目中，我们的目标是开发“超强”启动子，这将显著提高转基因的生产率，包括用于制造的蛋白质和抗体。

The production of recombinant proteins for medical research and healthcare is a rapidlyexpanding area of biotechnology. However, many of these proteins must be manufactured inexpensive mammalian systems to ensure correct protein folding, where the yield is typicallylow in comparison to bacterial systems (usually 100-1000-fold lower). In part this is due to theinferior strength of mammalian promoters (that control protein production) in comparison tobacterial counterparts.The strength of bacterial promoters is easy to predict, and the amount of protein produced isoften proportional to how similar the promoter used is to the ‘optimal’ consensus promoter. Inmammalian promoters, these same predictions cannot be made because the structures aremuch more complex, making it difficult to know which sections of the promoter mightenhance protein production. As a result, most mammalian expression systems are based onnaturally-occurring ‘wild-type’ promoters, which have actually evolved for physiologicalfunctions, not commercial protein manufacture. Our preliminary data show that significantlystronger promoters can be developed using our technology, with significant implications forprotein manufacture.At Oxford Genetics Ltd we have developed a ‘bioselection’ cloning system called‘Prometheus’ to screen large numbers (>4000) of recombinant mammalian promoters. Thetechnique allows a wide array of strong ‘wild-type’ mammalian promoters to be ‘shuffled’ andscreened to identify new composite promoters that produce more protein than the current goldstandards, CMV and CAG. Preliminary results are exciting, and in this project we aim todevelop ‘superstrength’ promoters that will dramatically improve the productivity oftransgenes, including proteins and antibodies for manufacture.