Enhancing Human Glycoprotein Hormones Production in CHO Cells Using Heterologous Beta-Chain Signal Peptides.

We studied the influence of heterologous signal peptides in the ß-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When the natural signal peptides of the ß-chains were replaced with the heterologous signal peptide of human serum albumin, cell productivity was increased 2-2.5 times for human luteinizing hormone, human chorionic gonadotropin, and human thyroid-stimulating hormone, but not for human follicle-stimulating hormone. No significant increase in cell productivity was observed for human azurocidin signal peptide and human glycoprotein hormone α-chain signal peptide. The used approach allows quick assessing the effect of heterologous signal peptides on the biosynthesis of heterodimeric proteins of various classes.

Targeted Knockout of the dhfr, glul, bak1, and bax Genes by the Multiplex Genome Editing in CHO Cells.

The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. Simultaneous editing of multiple genes makes it possible to obtain a cell line with the required genotype faster than several consecutive rounds of genomic knockout, but the probability of success is lower. Simultaneous editing of the dhfr, glul, bak1, and bax genes in the CHO S cells genome yielded 24 clones with signs of auxotrophy for thymidine and glutamine. Five of them turned out to be dhfr+/-, all five contained a knockout of one or two glul alleles. In one clone, 7 out of 8 target alleles were inactivated by a frameshift, and the second dhfr allele was partially inactivated by insertion of the GAA triplet, which reduced the enzyme activity 2.5 times. The probability of simultaneous knockout of both dhfr alleles increased to 50% when the genome was edited with a pair of guide RNAs directed to one exon of the dhfr gene.