| Compared with the microbial expression system, the mammalian cell expression system is more preferred to express recombinant proteins because of its advantages in the posttranslational modifications, such as glycosylation. At present, the mammalian cell-expressed recombinant protein drugs have occupied the major share of modern biological medicine market. However, the mammalian cell also has some drawbacks, such as apoptosis, low productivity and incomplete glycan structures compared with their natural forms, especially the terminal sialic acid deficience and mismatch, which reduce the production efficiency and biological activity of recombinant proteins. Therefore, in the future industry development, it is urgent to reengineer recombinant cell lines to improve their performance using genetic engineering and metabolic engineering approaches and so on.In previous studies, the silkworm hemolymph (SH) was found to inhibit apoptosis in both insect and human cells. The anti-apoptotic component was determined to be a group of structurally related proteins which consists of five isoforms including 30Kc6,30Kc12, 30Kc19,30Kc21 and 30Kc23 with molecular weights of approximately 30 kDa (30K proteins). Subsequently, the 30Kc6,30Kc12 and 30Kc19 genes were successfully cloned and expressed in Chinese hamster ovary (CHO) DG44 cells and their anti-apoptotic effects were examined. As a result, only the 30Kc6 gene exhibited potent anti-apoptotic activity. But in other studies the 30Kc19 gene was found to have significant influence on recombinant protein productivity and glycosylation. However, the mechanisms of 30Kc6 and 30Kc19 genes were not revealed yet, so it is concerned that whether they can be widely used to improve the performance of recombinant cell lines. Therefore, in this work, two CHO cell lines producing monoclonal antibody (mAb) and recombinant human Erythropoietin (EPO) were respectively transfected with the 30Kc6 gene and the 30Kc19 gene to examine the effects of 30Kc6 gene expression on apoptosis inhibition and the 30Kc19 gene expression on EPO productivity and glycosylation. Furthermore, the mechanisms of these two genes were also investigated in detail. Based on this work, it is hopefull that the 30K gene from Bombyx mori could be used to improve the performance of industrial recombinant CHO cell lines in the future.First, the 30Kc6 gene was transfected into CHO cells producing a chimeric anti-human CD20 mAb. Three stable cell lines with different expression levels of 30Kc6 protein and three stable negative control cell lines with vector alone were establiehed and then cultured in 6-well cell culture plates in adherent culture mode. It was found that the 30Kc6 gene expression significantly inhibited cell apoptosis and increased the specific mAb productivity (qmAb) by 1.3 folds. Consequently, the mAb production increased to 3.4 folds relative to the control. Subsequently, the mechanisms of 30Kc6 gene expression inhibiting cell apoptosis and increasing qmAb were investigated in detail. The results show that the 30Kc6 gene expression repressed the Bax protein translocation from cytosol to mitochondria and then reduced the Cytochrome c release from mitochondrial intermembrane space to cytosol, which resulted in a decrease in the activation of apoptosis executioner, Caspase-3. Thus, cell apoptosis was inhibited. On the other hand, the 30Kc6 gene expression significantly maintained the mitochondrial membrane potential (MMP) and the ATP generation. A high ATP level could enhance the activity of mammalian target of rapamycin (mTOR), which drives the translation initiation and elongation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). In the 30Kc6-expressing cell, both 4EBP1 and S6K1 were found to be phosphorylated at higher levels, indicating that the 30Kc6 gene expression significantly enhanced the translation process. Moreover, at the transcriptional level, the relative levels of heavy and light chain mRNAs increased only by 8.3% and 8.7%, respectively, indicating that the 30Kc6 gene expression did not significantly influence the transcription process. It is concluded that the 30Kc6 gene expression significantly increased qmAb because it maintained the ATP generation and consequently increased the mTOR activity, which enhanced the translation process.Hyperosmotic pressure, which can induce cell apoptosis, is a big problem always encountered in process development and optimization of the fed-batch culture. From this point, the established 50Kc6-expressing clones and the negative control clones were adapted to suspension culture, and the effect of 30Kc6 gene expression on hyperosmotic pressure-induced apoptosis was investigated. After the osmotic pressure was elevated from 310 (normal medium) to 410 mOsm/kg, apoptosis was analyzed by flow cytometry. As a consequence, the apoptotic rate of 30Kc6-expressing cell increased by 4.74% two days after osmotic pressure elevation, while the negative control cell increased by 15.45%, indicating that the hyperosmotic pressure-induced apoptosis was significantly inhibited by 30Kc6 gene expression. In addition, it was observed that in the hyperosmotic culture the qmAb of 50Kc6-expressing cell also significantly increased compared with that of the negative control cell. Overall, after osmotic pressure elevation, the 30Kc6 gene expression inhibited hyperosmotic pressure-induced apoptosis and enhanced qmAb, so that the antibody production significantly increased. To investigate the effects of 30Kc 19 gene and its mechanisms, both the gene expression and the recombinant protein addition strategies were employed. First, the 30Kc19 gene was transfected into CHO cells producing EPO, and three stable 30Kc 19-expressing clones and one negative control clone (with vector alone) with similar growth characteristics to the host cell were established. The clones were then cultured in 12-well cell culture plates in adherent culture mode. It was found that the EPO production and specific EPO productivity ( qEPO) increased by 102.6% and 146% with 30Kc19 gene expression, respectively. The sialic acid content was compared by two-dimensional electrophoresis (2-DE) and Western blot analysis. The result shows that the isoforms of EPO produced by the 50Kcr19-expressing cell obviously shifted from around pI6.5 to 4.8 relative to the control, indicating that the sialic acid content significantly increased. Regarding the mechanism of 30Kc19 gene expression enhancing qEPO, the MMP and ATP generation were found to be maintained by its expression. A high ATP level could enhance the translation process resulting in an increase in qEPO.Meanwhile, the 30Kc19 gene expression maintained sialyltransferase activity which increased the sialic acid content.Subsequently, the 30Kc19-expressing cell and the negative control cell were adapted to suspension culture, and the effects of 30Kc19 gene expression on EPO productivity and sialylation were further investigated. The result shows that the EPO production increased to 2.5 folds compared with the control. The N-linked glycans (N-glycans) including glycan structures, sialic acid and fucose contents, were analyzed using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). It was found that the N-glycans produced by the 30Kc19-expressing cell consisted of bi-, tri-, tetra- and penta-antennary branching (35.12%,18.12%,33.14% and 13.62%, respectively), while the negative control cell predominantly produced the N-glycans with bi-antennary branching (70.00%). In addition,52.89% of the N-glycans produced by the 30Kc19-expressing cell was terminally sialylated and 76.83% was fucosylated. However, no obvious sialylated N-glycan was detectable for the negative control cell and only 61.45% of the N-glycans was fucosylated. These results indicate that the N-glycans produced by the 30Kc19-expressing cell formed more complex structures, and the sialic acid and fucose contents significantly increased.Finally, the 30Kc19 protein was expressed and purified from Escherichia coli, and then added into the serum-free suspension culture medium with a concentration of 0.2 mg/ml to investigate its effects on EPO productivity and sialylation. The results show that the EPO production increased by 41.6% and the isoforms of EPO significantly shifted from around pI 6.2 to 4.6 compared with the control, indicating that the sialylation also enhanced.These results demonstrate that both the 30Kc19 gene expression and the 30Kc19 protein addition can enhance EPO productivity and sialylation in the suspension culture. In contrast, the gene expression method is more effective.Based on the above research work, the 30Kc6 gene expression was found to significantly inhibit cell apoptosis, and the 30Kc19 gene expression significantly enhanced recombinant protein productivity and glycosylation. Moreover, their mechanisms were also revealed. In conclusion, reengineering recombinant CHO cell lines with the 30K gene from Bombyx mori significantly inhibits cell apoptosis and increases recombinant protein production and glycosylation. Thus, the 30K gene is a novel and effective gene with great potential for improving the performance of recombinant cell lines.
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