There is a growing demand for rapid, large-scale production of recombinant proteins, either as biotherapeutics or as research tools. The use of mammalian expression systems for the production of recombinant targets has significantly increased, due to their ability to perform complex post-translational modifications. However, certain recombinant proteins are deemed 'difficult to express' in mammalian expression systems and the unpredictability of production has significant consequences on drug development processes. To this effect, published examples of 'difficult to express' proteins have reported 'bottlenecks' at different stages along the protein expression pathway. As a result, separate protein-specific cell engineering strategies have been employed to overcome such limitations. No universal approach has been defined to overcome poor recombinant protein production. This Thesis has investigated the molecular mechanisms that restrict expression of recombinant proteins of extensive sequence similarity. The limiting step(s) that prevent successful production of a panel of recombinant targets have been defined by systematic screening of cellular processes along the protein expression pathway. It was found that the processes that restrict efficient protein production were of a protein-specific nature. Further, a protein engineering strategy was developed that allowed successful secretion of 'difficult to express' targets limited in their post-translational processing after initial processing in the endoplasmic reticulum. Screening of amino acid sequences using a computational approach reported on the predictability of protein secretion and identified problematic sequence features that may limit secretion. Computational analyses revealed that increased abundance of positively-charged or hydrophobic surface regions was associated with poor or no protein secretion. Engineering of these problematic sequence features in a model 'difficult' recombinant target resulted in successful secretion. Therefore, application of computational screening and re-engineering of unfavourable sequence features prior to expression in mammalian cells offers the potential to overcome challenges in the production of 'difficult to express' recombinant targets.A further theme focused on identification of cell engineering for creation of robust host cell lines for the production of recombinant proteins. Transcriptomic profiling revealed gene expression response distinctions between Chinese Hamster Ovary cells that expressed retained and secreted proteins. That study also highlighted significant gene expression changes between cells in early and later cell growth phase. Transcripts that were significantly differentially expressed were linked to endoplasmic reticulum stress and/or unfolded protein response signalling and cell cycle canonical pathways. Targeting of upstream regulators within these pathways acts as a potential cell engineering approach to enhance recombinant protein production.