Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.

A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks.

Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.

RAD51 is involved in repair of damage associated with DNA replication in mammalian cells.

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.

Functional properties of Kch, a prokaryotic homologue of eukaryotic potassium channels.

To test the hypothesis that the Kch gene of Escherichia coli encodes a potassium channel, we have transformed E. coli with an expression vector containing the Kch sequence and observed the effect of over-expression of Kch on E. coli. We found that: (i) over-expression of Kch is toxic to E. coli, but the toxicity could be prevented by supplementing the growth medium with K(+), Rb(+), and NH(4)(+), but not Na(+), consistent with the properties of a potassium selective pore; (ii) Cs(+), a blocker of potassium channels, rescues the growth of Kch over-expressing cells; and (iii) when the putative pore-forming region of Kch, containing the signature sequence, was replaced with the corresponding region of the eukaryotic Shaker potassium channel, and the resultant construct expressed in E. coli, the cells became critically dependent on K(+) supply for survival. These data are consistent with the proposed function of Kch, i.e., K(+) conduction.

Defects in homologous recombination repair in mismatch-repair-deficient tumour cell lines.

Loss of mismatch repair (MMR) leads to a complex mutator phenotype that appears to drive the development of a subset of colon cancers. Here we show that MMR-deficient tumour cell lines are highly sensitive to the toxic effects of thymidine relative to MMR-proficient lines. This sensitivity was not a direct consequence of MMR deficiency or alterations of DNA precursor metabolism. Instead, MMR-defective tumour cell lines are also defective in homologous recombination repair (HRR) induced by DNA double-strand breaks. Furthermore, a frameshift mutation of the human RAD51 paralog XRCC2 found in the MMR-deficient uterine tumour cell line SKUT-1 can confer thymidine sensitivity when introduced into a MMR-proficient line. Like other cells with defective XRCC2, SKUT-1 is sensitive to mitomycin C, and MMR-proficient cells expressing the mutant XRCC2 allele become more sensitive to this agent. These data suggest that the thymidine sensitivity of MMR-deficient tumour cell lines may be a consequence of defects in the HRR pathway. The increased thymidine sensitivity and the loss of an important pathway for the repair of DNA double-strand breaks create new opportunities for therapies directed specifically against this subset of tumours.