The Gly is the “naturally preferred” amino acid at this position and is present in ~50% of human antibody sequences with Ala and Ser equally contributing to the other 50%. They suggested the increase in protein expression was due to a combination of increased mRNA half-life and improved fidelity of protein folding and post-translational events at 32 ☌.Ī pair of IgG 4 antibodies was previously identified whereby an Ala to Gly mutation at Kabat position 49 resulted in a four-fold decrease in transient CHO expression. Recently the Smales’ group showed that cap-dependent mRNA translation is not severely attenuated in CHOK1 cells when the culture temperature is shifted from 37 ☌ to 32 ☌. To explain why the opposite effect is often observed, reports suggest increased RNA half-life, increased transcription of certain target genes through specific binding sites within their promoter regions, alternative mRNA splicing, and CSP-mediated Internal Ribosome Entry Segments (IRESs) in certain mRNAs may be responsible for increased gene expression at sub-physiological temperatures. Intuitively, the repression of the gene expression machinery should decrease exogenous protein production. The general response of mammalian cells to cold is thought to involve a combination of responses regulated by cold-shock proteins (CSPs) including reduced metabolism, cell-cycle arrest, predominately in the G 1 phase, increased viability for prolonged periods of time, and transcriptional/translational attenuation. We propose that the effect of reduced culture temperature on expression is protein-dependent protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. The difference in specific productivity was reflected in the amounts of heavy- and light-chain mRNA. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 ☌ compared to 37 ☌. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase.
Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells.
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