Enhancing global and mRNA specific translation for improved recombinant protein expression in in vitro cultured mammalian cells

目前正在开发的许多新药都是基于蛋白质而不是传统的小分子(例如抗生素)。其中一种特别具有挑战性的蛋白质分子是抗体，例如赫赛汀。这些蛋白质药物是通过在特定条件下培养的细胞生产出来的，用于治疗癌症等疾病。这样做的一个问题是，我们用来制造治疗用蛋白质的细胞并不像我们希望的那样有效，因此我们可能无法生产足够的这些药物，而且对它们的成本和需求很高。蛋白质合成是将细胞中遗传物质中的信息，即DNA，通过中间底物mRNA转化为蛋白质的过程。要合成蛋白质，信使核糖核酸必须与称为核糖体的大型复合体相互作用，核糖体由RNA和蛋白质组成。核糖体能够破译信使核糖核酸中的遗传信息，并进行蛋白质的合成。有两种不同的机制可以使mRNAs与核糖体相互作用。最常见的机制需要蛋白质复合体与mRNA的5‘端结合，然后这个复合体招募核糖体。然而，某些mRNA包含不编码蛋白质片段的5‘区(称为非翻译区；UTRs)，这些RNA序列包含形成复杂RNA结构所需的信息。这些RNA结构允许核糖体被募集到mRNA中，通常距离5‘端相当远，因此这种核糖体募集的方法被称为核糖体内部进入。有趣的是，使用内部核糖体进入的信息通常编码在细胞应激情况下使用的蛋白质，包括在温度降低(冷休克)下使用的蛋白质。这一信息具有工业意义，因为当细胞在培养后期受到压力时，以及在发酵过程中通常引起的冷休克，会阻碍商业价值蛋白质(例如抗体)的生产。我们的目标是利用在冷休克期间翻译活跃的mRNAs的5‘UTRs来促进对工业重要的蛋白质的生产。实现这一点非常重要，因为预计随着越来越多的蛋白质药物正在开发，我们将缺乏能力生产足够大的数量来满足大多数人对这些新药的需求，而不是那些能够负担得起目前仍然贵得令人望而却步但非常有效的药物的人。

Many of the new drugs currently under development are based upon proteins rather than traditional small molecules (e.g. antibiotics). One of the type of protein molecules that is particularly challenging to make are antibodies e.g. herceptin. These protein drugs are produced for the treatment of diseases such as cancer by cells kept in culture under defined conditions. One problem with this is that the cells we use to make proteins for therapeutic uses are not as efficient as we would like them to be and therefore we may not be able to produce enough of these drugs and the cost and demand for them is high. Protein synthesis is the process by which the information in the genetic material in the cell, DNA is converted via an intermediary substrate mRNA, into proteins. For proteins to be synthesised the mRNA must interact with a large complex called the ribosome which consists of RNAs and proteins. Ribosomes are able to decode the genetic information that is held in the mRNA and carry out the synthesis of the proteins. There are two distinct mechanisms by which mRNAs can interact with the ribosomes. The most common mechanism requires the binding of a protein complex to the 5' end of the mRNA and this complex then recruits the ribosome. However, certain mRNAs contain 5' regions that do not code for sections of proteins (termed untranslated regions; UTRs) and these sequences of RNA harbour the information that is required to form a complex RNA structure. These RNA structures allow the ribosome to be recruited to the mRNA generally a considerable distance from the 5' end and so this method of ribosome recruitment has been termed internal ribosome entry. Interestingly, messages that use internal ribosome entry generally encode proteins that are used under situations of cell stress including under temperature reduction (cold-shock). This information is of industrial relevance since the production of commercially valuable proteins (e.g. antibodies) is hindered when cells become stressed later in culture and by the cold-shock that is commonly induced during fermentation. We aim to use the 5' UTRs of mRNAs that are translationally active during cold-shock to enhance the production of proteins that are important to industry. Achieving this is very important as it is expected that with an increasing number of protein 'drugs' being developed we will lack the capability of producing large enough amounts to meet the required demand for these new drugs for the majority, as opposed to for those who can afford what must currently remain prohibitively expensive, but very effective, medicines.