Abstract
BackgroundExpression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency.ResultsAfter a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture.ConclusionsNovel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0145-9) contains supplementary material, which is available to authorized users.
Highlights
Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity
Design of mammalian expression vectors with attenuated dhfr selection marker In our previous study, we have demonstrated that selection marker attenuation with destabilizing sequences to reduce the transcript and/or protein levels of the selection marker gene, can improve the production of the recombinant gene of interest upon selection and MTX amplification [31]
Codon deoptimized dhfr has been used by Westwood AD et al [35] to improve recombinant protein yield in serumcontaining Chinese hamster ovary (CHO) cell culture that were not exposed to MTX, though a different sequence is used in our study
Summary
Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. The titers of biopharmaceutical production from CHO cells have achieved gram per liter range and this 100 fold improvement since 1980s can be attributed to advances in bioprocess development, media development and cell line development. While many of these bioprocess and culture media improvements were kept as trade secrets [7], cell line development technologies like expression vector engineering, cell line engineering and clone screening technologies were extensively reviewed [1,7,8,9,10]. Together with the discovery of genome wide editing tools like zinc finger nucleases, transcription activator-like effector nucleases and meganucleases, more of these genes can be validated for their roles in biopharmaceuticals production [19,20,21]
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