Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

No abstract

No abstract

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.