Utilizing a regulated targeted integration cell line development approach to systematically investigate what makes an antibody difficult to express.

Chinese hamster ovary (CHO) cells are conventionally used to generate therapeutic cell lines via random integration (RI), where desired transgenes are stably integrated into the genome. Targeted integration (TI) approaches, which involve integration of a transgene into a specific locus in the genome, are increasingly utilized for CHO cell line development (CLD) in recent years. None of these CLD approaches, however, are suitable for expression of toxic or difficult-to-express molecules, or for determining the underlying causes for poor expression of some molecules. Here we introduce a regulated target integration (RTI) system, where the desired transgene is integrated into a specific locus and transcribed under a regulated promoter. This system was used to determine the underlying causes of low protein expression for a difficult-to-express antibody (mAb-A). Interestingly, we observed that both antibody heavy chain (HC) and light chain (LC) subunits of mAb-A independently contributed to its low expression. Analysis of RTI cell lines also revealed that while mAb-A LC triggered accumulation of intracellular BiP, its HC displayed impaired degradation and clearance. RTI pools, generated by swapping the WT or point-mutant versions of difficult-to-express antibody HC and LC with that of an average antibody, were instrumental in understanding the contribution of HC and LC subunits to the overall antibody expression. The ability to selectively turn off the expression of a target transgene in an RTI system could help to directly link expression of a transgene to an observed adverse effect. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2772, 2019.

Posttranscriptional regulation of HO expression by the Mkt1-Pbp1 complex.

Cells of budding yeast give rise to mother and daughter cells, which differ in that only mother cells express the HO endonuclease gene and are thereby able to switch mating types. In this study, we identified the MKT1 gene as a positive regulator of HO expression. The MKT1 gene encodes a protein with two domains, XPG-N and XPG-I, which are conserved among a family of nucleases, including human XPG endonuclease. Loss of MKT1 had little effect on HO mRNA levels but resulted in decreased protein levels. This decrease was dependent on the 3' untranslated region of the HO transcript. We screened for proteins that associate with Mkt1 and isolated Pbp1, a protein that is known to associate with Pab1, a poly(A)-binding protein. Loss of PBP1 resembles an mkt1 Delta deletion, causing decreased expression of HO at the posttranscriptional level. Mkt1 and Pbp1 cosedimented with polysomes in sucrose gradients, with Mkt1 distribution in the polysomes dependent on Pbp1, but not vice versa. These observations suggest that a complex of Mkt1 and Pbp1 regulates the translation of HO mRNA.

The Khd1 protein, which has three KH RNA-binding motifs, is required for proper localization of ASH1 mRNA in yeast.

RNA localization is a widespread mechanism for achieving localized protein synthesis. In Saccharomyces cerevisiae, Ash1 is a specific repressor of transcription that localizes asymmetrically to the daughter cell nucleus through the localization of ASH1 mRNA to the distal tip of the daughter cell. This localization depends on the actin cytoskeleton and five She proteins, one of which is a type V myosin motor, Myo4. We show here that a novel RNA-binding protein, Khd1 (KH-domain protein 1), is required for efficient localization of ASH1 mRNA to the distal tip of the daughter cell. Visualization of ASH1 mRNA in vivo using GFP-tagged RNA demonstrated that Khd1 associates with the N element, a cis-acting localization sequence within the ASH1 mRNA. Co-immunoprecipitation studies also indicated that Khd1 associates with ASH1 mRNA through the N element. A khd1Delta mutation exacerbates the phenotype of a weak myo4 mutation, whereas overexpression of KHD1 decreases the concentration of Ash1 protein and restores HO expression to she mutants. These results suggest that Khd1 may function in the linkage between ASH1 mRNA localization and its translation.