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

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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.