Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.     

Protective Effect of the Silkworm Protein 30Kc6 on Human Vascular Endothelial Cells Damaged by Oxidized Low Density Lipoprotein (Ox-LDL)

Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs). In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL) and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS) generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK), especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC). Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti-apoptotic mechanism of the 30K family proteins, but also provide important information on prevention and treatment of human cardiovascular diseases.     

Enhancement of cell growth and viability of CHO cells in serum-free media by 30Kc6 gene expression

Many attempts have been made to develop a serum-free medium on account of the problems caused by serum in mammalian cell culture. However, serum deprivation inhibits cell growth and induces apoptosis. Moreover, adapting host cells to the serum-free medium is difficult and time-consuming. In a previous study, the anti-apoptotic 30K proteins were identified from silkworm hemolymph, which suggests that the 30K genes coding for the anti-apoptotic compound can be used for the anti-apoptosis engineering of mammalian cells. In this study, the 30K genes (30Kc6, 30Kc19, and 30Kc123) were introduced to DG44 CHO cells, which are the mammalian cell line most commonly used by industry for the production of biopharmaceuticals, in order to make them resistant to the apoptosis induced by serum deprivation. Among the 30K genes, the 30Kc6 gene exhibited the highest apoptosis-inhibition activity. When the 30Kc6-expressing cells cultivated in the serum-containing medium were transferred directly to commercially available serum-free media, 30Kc6 expression increased the viable cell density by four-fold through inhibiting serum deprivation-induced apoptosis.     

Stabilization of enzymes by the recombinant 30Kc19 protein

In previous studies, we reported that the 30K protein originating from the silkworm inhibited apoptosis in mammalian cells. In this work, we demonstrated that the 30Kc19 protein, which is most abundant 30K protein in silkworm hemolymph, also enhanced enzyme stability. When the recombinant 30Kc19 protein was supplemented into distilled-deionized water containing alkaline phosphatase or horseradish peroxidase, deactivation of both enzymes induced by non-buffered DDW was significantly suppressed. The increase in enzyme stability due to the presence of 30Kc19 was similar to that observed for bovine serum albumin, which is commonly used in conventional enzyme reactions. The decrease in enzyme activity due to long-term storage in different buffer systems was also inhibited by 30Kc19. The 30Kc19 protein structure was shown to play a vital role in stabilizing the enzyme. These results imply that the recombinant 30Kc19 protein hold promise for use as an additive to increase or maintain enzyme activity.     

Inhibition of HeLa Cell apoptosis by storage-protein 2

Apoptosis is a barrier to maintaining high viable cell densities in animal cell culture. Silkworm hemolymph and its 30K protein have been reported to exhibit anti-apoptotic activity in various mammalian and insect cell systems. The 30K protein is thermally unstable at temperatures higher than 60 degrees C; however, the silkworm hemolymph heat-treated at 70-80 degrees C still exhibited anti-apoptotic activity. This indicates that silkworm hemolymph contains another anti-apoptotic compound other than 30K protein. In this article, the anti-apoptotic molecule other than 30K protein was found from the silkworm hemolymph and identified. This molecule was storage-protein 2 (SP2), which has no homology with any known anti-apoptotic protein. This molecule was heat-stable up to 80 degrees C, while 30K protein lost its activity at temperatures higher than 60 degrees C. When apoptosis was induced by staurosporine in HeLa cells, SP2 protein suppressed nuclear fragmentation and apoptotic body formation. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. Inhibition of apoptosis by the SP2 protein would lead to the minimization of cell death during commercial mammalian cell culture.     

Inhibition of apoptosis by a Bombyx mori gene

An apoptosis-inhibiting component of silkworm hemolymph, isolated and characterized in our previous study, showed 95% N-terminal amino acid sequence homology with one of the 30K proteins, a group of structurally related proteins. The 30K protein was expressed in mammalian HEK293 cells and CHOK1 cells by transfection with 30Kc6. The expression of 30Kc6 inhibited apoptosis comparably to that of whole silkworm hemolymph, indicating that both intracellular expression and external supplementation inhibited apoptosis. The expression of 30Kc6 resulted in lower intracellular activity for caspase 3. However, the results of in vitro assay of caspase 3 show that the 30Kc6 protein does not inhibit caspase 3 activity. This indicates that the 30Kc6 protein inhibits the apoptosis by working in a further upstream event than caspase 3 activation.     

Enhancement of recombinant human EPO production and sialylation in chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression

Productivity and sialylation are two important factors for the production of recombinant glycoproteins in mammalian cell culture. In our previous study, we found that silkworm hemolymph increased the sialylation of recombinant secreted human placental alkaline phosphatase in the insect cells, promoted the transfer of sialic acids onto the glycoprotein oligosaccharides in an in vitro asialofetuin sialylation system, and enhanced recombinant protein production in the Chinese hamster ovary (CHO) cells. These beneficial effects were mainly due to the 30K proteins, which consist of five isoforms. Among the 30K proteins, 30Kc19 was determined to be the major component. In this study, the 30Kc19 gene was introduced into a CHO cell line producing recombinant human erythropoietin, and its effects on productivity and sialylation were investigated. The transient expression of 30Kc19 significantly improved the production and sialylation of EPO. A stable cell line containing 30Kc19 was also established to investigate the effect of 30Kc19 gene expression. The stable expression of 30Kc19 increased the production and sialylation by 102.6% and 87.1%, respectively. The enhanced productivity from 30Kc19 expression is believed to occur because the 30Kc19 protein suppresses the loss of mitochondrial membrane potential and consequently improves the generation of intracellular ATP. In addition, the positive effect of 30Kc19 expression on sialylation is believed to be due to its ability to maintain sialyltransferase activity. In conclusion, 30Kc19 expression is a novel approach to improve the production and sialylation of recombinant glycoproteins in CHO cells.     

Anti-apoptosis engineering

An increased understanding of apoptosis makes anti-apoptosis engineering possible, which is an approach used to inhibit apoptosis for the purpose of therapeutic, or industrial applications in the treatment of the diseases associated with increased apoptosis, or to improve the productivity of animal cell cultures, respectively. Some known anti-apoptotic proteins are the Bcl-2 family, IAP (inhibitor of apoptosis) and Hsps (heat shock proteins), with which anti-apoptosis engineering has progressed. This article reviews anti-apoptosis engineering using known anti-apoptotic compounds, and introduces a 30 K protein, isolated from silkworm hemolymph, as a novel anti-apoptotic protein, that shows no homology with other known anti-apoptotic proteins. The regulation of apoptosis, using anti-apoptotic proteins and genes originating from the silkworm,Bombyx mori, may provide a new strategy in this field.     

Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells

We have previously shown that silkworm hemolymph exhibits anti-apoptotic activity against baculovirus-induced Sf9 cell apoptosis. In this study, using various chemical inducers, such as actinomycin D, camptothecin, and staurosporine, we found that silkworm hemolymph inhibits insect cell apoptosis induced not only by baculovirus but also by chemical inducers. This indicates that silkworm hemolymph contains anti-apoptotic components that work directly in insect cell apoptosis without any booster expression of baculoviral genes. With the analysis of Sf-caspase-1 activity, it was found that the inhibitory effect of silkworm hemolymph works in a further upstream step than the Sf-caspase-1 activation step.     

Anti-oxidative effects of silkworm storage protein 1 in HeLa cell

Silkworm hemolymph contains unique proteins that exhibit anti-apoptotic activity in mammalian cells. Among them, 30 K protein, which is one of the major anti-apoptotic molecules in silkworm hemolymph, has been well investigated. However, little is known about the biological functions of storage protein 1 (SP1), another main protein in silkworm hemolymph. In this study, the anti-apoptotic and anti-oxidative activities of SP1 were analyzed. A stable cell line expressing SP1 was constructed, which showed strong anti-apoptotic effect induced by staurosporine treatment. In addition, the cell line exhibited resistance to oxidative stress caused by hydrogen peroxide. For practical applications of SP1, recombinant SP1 was produced in Escherichia coli, and the supplementation of recombinant SP1 into culture medium exhibited anti-apoptotic and anti-oxidative activities. In addition, SP1 was found to be a cell-penetrating protein and localized in the cytosol as well as on the plasma membrane. The findings showed that SP1 itself is not an anti-oxidant; rather, it mediates intracellular anti-oxidative activity. In conclusion, the cellular resistance of SP1 to apoptosis and oxidative stress will provide a new strategy that could be utilized in the bio-industry for the production of biologics as well as for the development of anti-aging cosmetics.     

Inhibition of human cell apoptosis by silkworm hemolymph

Many studies on preventing apoptosis have been carried out from the viewpoint of anti-apoptotic cloned-gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus-induced insect cell apoptosis. Using the vaccinia virus-HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus-induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture.     

Enhancement of therapeutic monoclonal antibody production in CHO cells using 30Kc6 gene

Over-expression of anti-apoptotic cloned-genes is a widely used strategy for inhibiting apoptosis in mammalian cell culture. In our previous study, we reported Bombyx mori 30K gene improved the production of recombinant proteins in Chinese hamster ovary (CHO) cells. In this study, we reengineered the CHO cells with the 30Kc6 gene and 30Kc19 gene for the production of a therapeutic monoclonal antibody (mAb) directed against the glycoprotein receptor of human platelets. After the medium was changed from serum containing one to serum-free one, expression of 30Kc6 in CHO cells increased the cell viability by 40.8% in 4 days and mAb production by 2.3-fold in 5 days. However, no significant changes in cell viability and mAb production were observed for the cells expressing 30Kc19. In the case of the cells expressing 30Kc6, the specific production rate was also improved. The expression of the 30Kc6 gene increased the cell viability and productivity because it maintained the mitochondrial membrane potential (MMP) and reduced the downstream cascade responses for apoptosis. These results indicate that 30Kc6 outperformed 30Kc19 in terms of cell death-protective capability and the production of monoclonal antibodies in CHO cells.     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

Increase of 30K protein in identified motoneurons by hemolymph results in inhibition of programmed cell death in silkworm, Bombyx mori (Lepidoptera, Bombycidae)

This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph.     

Exploring Binding Mechanisms between Curcumin and Silkworm 30Kc19 Protein Using Spectroscopic Analyses and Computational Simulations

The curry compound, curcumin exerts multiple health-promotive functions; however, its poor solubility and stability limits its biological applications. In this study, we illuminate intermolecular binding mechanisms in the nano-sized complex of curcumin with silkworm protein, 30Kc19. The intrinsic fluorescence of 30Kc19 was gradually quenched by the increase of curcumin concentrations, which demonstrates molecule-molecule complexations mediated by the fluorophore amino acid residues (Tyr, Trp) in the protein. The fluorescence quenching showed that the binding occurred at 1:1 molar ratio with binding constant of 3.28 × 10⁴ M^-1. The results from scanning electron microscopy and dynamic light scattering indicate that the complexes were formed with cubicle shapes and sizes of 200–250 nm at pH 8.0 (zeta-potential < ?20 mV). Along with Fourier transform infrared analysis, computational studies of protein-ligand docking simulation suggest a mechanism that curcumin and 30Kc19 forms complexes through specific amino acid residues (Trp174, Trp180, and Trp225) with minimum binding distance (4 Å). The complexation of curcumin with 30Kc19 protein effectively suppressed the degradation of curcumin over 10 h and improved its antioxidant activity up to 30%. These findings suggest an application of 30Kc19 for the delivery of waterinsoluble bioactive medicines.     

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K _m values of the enzyme for 1,3-propanediol and NAD⁺ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.     

Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.     

毕赤酵母甲酸脱氢酶在大肠杆菌中的高表达及纯化

用PCR方法从毕赤酵母(Pichia pastoris)基因组DNA扩增甲酸脱氢酶(FDH)基因,通过定点突变密码子TAG(649-651位碱基)为GAG,突变后的基因片段插入表达载体pET-22b( )构建质粒pET-FDH,转化E．coli BL21(DE3)。基因工程菌在IPTG诱导下高效表达可溶性的FDH融合蛋白,蛋白的表达量占基因工程菌株总蛋白的30%。工程菌破壁上清液采用一步亲和层析分离,得到比活力为6．45U/mg的重组FDH。     

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14?%, respectively), while the control cells produced predominantly bi-antennary branching (70?%). About 53?% of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77?%, whereas only 61?% of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6?% after the addition of 0.2?mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.     

Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis

The effect of silkworm hemolymph on baculovirus-induced insect cell apoptosis was investigated. The addition of silkworm hemolymph into the culture medium either before or during the baculovirus infection increased the host cell longevity; however, its addition after the infection was less effective. This can be explained by the higher transfer rate of silkworm hemolymph which is caused by endocytosis during the virus internalization step. The delayed cell death due to silkworm hemolymph was not caused by an inhibition of the virus attachment and internalization steps. The apoptosis was analyzed using DNA fragmentation and TUNEL assays, and the resulting data confirm that silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.