Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells.

Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.

Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins.

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, 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 for metabolic engineering of HEK293 and CHO.

Studying Nestin and its Interrelationship with Cdk5.

Current research utilizes the specific expression pattern of intermediate filaments (IF) for identifying cellular state and origin, as well as for the purpose of disease diagnosis. Nestin is commonly utilized as a specific marker and driver for CNS progenitor cell types, but in addition, nestin can be found in several mesenchymal progenitor cells, and it is constitutively expressed in a few restricted locations, such as muscle neuromuscular junctions and kidney podocytes. Alike most other members of the IF protein family, nestin filaments are dynamic, constantly being remodeled through posttranslational modifications, which alter the solubility, protein levels, and signaling capacity of the nestin filaments. Through its interactions with kinases and other signaling executors, resulting in a complex and bidirectional regulation of cell signaling events, nestin has the potential to determine whether cells divide, differentiate, migrate, or stay in place. In this review, the broad and similar roles of IFs as dynamic signaling scaffolds, is exemplified by observations of nestin functions and its interaction with the cyclin- dependent kinase 5, the atypical kinase in the family of cyclin-dependent kinases.