Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster ovary cells.

BACKGROUND: 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. 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. RESULTS: After 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. CONCLUSIONS: Novel 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.

Constitutive overexpression of asm2 and asm39 increases AP-3 production in the actinomycete Actinosynnema pretiosum.

Constitutive overexpression of regulators in the ansamitocin biosynthetic cluster of Actinosynnema pretiosum was investigated as a strategy to increase the production of ansamitocin-P3 (AP-3), a clinically promising chemotherapeutic agent. Putative transcriptional regulators asm2, asm29, and asm34 as well as the putative regulatory protein asm39 were cloned into a single-site integrative vector and a multicopy replicative vector, pAP40 and pREP, respectively, and then transformed into A. pretiosum. Transformants overexpressing asm2 and asm39 in pREP showed an increase in ansamitocin production (1.3-fold over parental levels) in a bioassay screen. In shake-flask fermentations, the asm2 and asm39 overexpression transformants attained a maximum AP-3 titer of 33 and 52 mg/l, respectively, which were 1.6- and 2.5-fold higher than the blank vector control. The increase in AP-3 production for the asm2 overexpression transformant was unexpected, since prior reports suggested that Asm2 was a transcriptional repressor. The increase in production appeared to be dependent on the high expression levels achieved with the replicative vector, which may have disrupted the normal function of Asm2. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) confirmed that asm2 and asm39 transcription levels were significantly higher in the transformants relative to the control, suggesting that the yield improvement was due to the transformed plasmids. This study demonstrates that deregulated overexpression of regulatory genes is a feasible strategy to increase AP-3 production in A. pretiosum.