RESUMEN
We report a simple and effective means to increase the biosynthetic capacity of host CHO cells. Lonza proprietary CHOK1SV® cells were evolved by serial sub-culture for over 150 generations at 32 °C. During this period the specific proliferation rate of hypothermic cells gradually recovered to become comparable to that of cells routinely maintained at 37 °C. Cold-adapted cell populations exhibited (1) a significantly increased volume and biomass content (exemplified by total RNA and protein), (2) increased mitochondrial function, (3) an increased antioxidant capacity, (4) altered central metabolism, (5) increased transient and stable productivity of a model IgG4 monoclonal antibody and Fc-fusion protein, and (6) unaffected recombinant protein N-glycan processing. This phenotypic transformation was associated with significant genome-scale changes in both karyotype and the relative abundance of thousands of cellular mRNAs across numerous functional groups. Taken together, these observations provide evidence of coordinated cellular adaptations to sub-physiological temperature. These data reveal the extreme genomic/functional plasticity of CHO cells, and that directed evolution is a viable genome-scale cell engineering strategy that can be exploited to create host cells with an increased cellular capacity for recombinant protein production.
Asunto(s)
Cricetulus , Cricetinae , Animales , Temperatura , Células CHO , Biomasa , Proteínas RecombinantesRESUMEN
Macrocyclic peptides have promising therapeutic potential but the scaling up of their chemical synthesis is challenging. The cyanobactin macrocyclase PatGmac is an efficient tool for production but is limited to substrates containing 6-11 amino acids and at least one thiazoline or proline. Here we report a new cyanobactin macrocyclase that can cyclize longer peptide substrates and those not containing proline/thiazoline and thus allows exploring a wider chemical diversity.