Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

Abstract

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels

“…Treatment of siRNA depleted NeoR mRNA 87-92% resulting in an increase in both VCD and titer. 13 Fomina-Yadlin also showed strong correlation between mRNA levels and qP for a recombinant Fc-fusion protein and up to 45% of total cellular mRNA levels encoded the recombinant transcript for cultures treated with chemicals that induce gene expression. 24 We did not observe substantial differences in growth during production phase between pools selected while constitutively expressing recombinant protein compared to those only expressing recombinant protein during production phase (Figure 5b and Figure S3b).…”

“…In that example, the NeoR mRNA accounted for 5.63% of translated mRNAs on Day 3 and 5.41% on Day 6. Treatment of siRNA depleted NeoR mRNA 87–92% resulting in an increase in both VCD and titer . Fomina‐Yadlin also showed strong correlation between mRNA levels and qP for a recombinant Fc‐fusion protein and up to 45% of total cellular mRNA levels encoded the recombinant transcript for cultures treated with chemicals that induce gene expression …”

“…In mammalian cells, there tends to be a modest correlation between recombinant protein expression and growth rate indicating that metabolic burden also plays a role in limiting cellular productivity in mammalian expression systems . Kallehauge et al showed that reducing expression of a selectable marker using RNAi resulted in increased growth rate . One strategy to mitigate the impact of metabolic burden routinely used in microbial systems is to use inducible expression systems separating growth and expression phases therefore limiting the impact of metabolic burden to only the production phase of the culture when cell growth is not necessarily desirable.…”

“…[9][10][11][12] Kallehauge et al showed that reducing expression of a selectable marker using RNAi resulted in increased growth rate. 13 One strategy to mitigate the impact of metabolic burden routinely used in microbial systems is to use inducible expression systems separating growth and expression phases therefore limiting the impact of metabolic burden to only the production phase of the culture when cell growth is not necessarily desirable.…”

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis

“…On-the-other-hand, feeding of cells resulted in an increase in global translational activity and an increase in specific heavy and light chain translation [25]. The third found that recombinant mRNAs are translated as efficiently as host cell transcripts and occupied up to 15% of total ribosome capacity [26]. Collectively these studies conclude that a better understanding of global and target specific translational efficiency and reprogramming in CHO cells could aid in the development of feeding strategies to prolong growth and recombinant protein production and identify targets for cell engineering strategies to prolong or enhance recombinant gene synthesis.…”