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

用于医学研究和保健的重组蛋白质的生产是生物技术的一个快速扩展的领域。然而，这些蛋白质中的许多必须在廉价的哺乳动物系统中制造以确保正确的蛋白质折叠，其中与细菌系统相比，产量通常较低（通常低100-1000倍）。这在一定程度上是由于哺乳动物启动子（控制蛋白质产生）的强度不如细菌。细菌启动子的强度很容易预测，产生的蛋白质量通常与所用启动子与“最佳”共有启动子的相似程度成正比。在哺乳动物的启动子中，不能做出同样的预测，因为结构要复杂得多，很难知道启动子的哪些部分可能会增强蛋白质的产生。因此，大多数哺乳动物表达系统都是基于天然存在的“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，而不是为了商业化的蛋白质生产。我们的初步数据表明，使用我们的技术可以开发出明显更强的启动子，这对蛋白质生产具有重要意义。在牛津遗传学有限公司，我们开发了一种名为“普罗米修斯”的“生物选择”克隆系统，用于筛选大量（>4000）重组哺乳动物启动子。该技术允许一系列强大的“野生型”哺乳动物启动子进行“改组”和筛选，以确定新的复合启动子，其产生的蛋白质比目前的金标准CMV和CAG更多。初步的结果是令人兴奋的，在这个项目中，我们的目标是开发“超强”启动子，这将大大提高转基因的生产力，包括蛋白质和抗体的生产。

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.