Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.     

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.     

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.     

Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.     

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.     

Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering

Production of complex recombinant proteins requires the culture of mammalian cells in bioreactors. Inherent in these cultures is the problem of cell death, which can result from nutrient depletion, byproduct accumulation, and other bioreactor stresses which signal the cell to die through apoptosis, or programmed cell death. Apoptosis is a highly regulated pathway of both pro- and anti-apoptotic proteins that promote cell survival or death, and cell engineering efforts to inhibit the apoptosis pathway have led to increased culture viability and recombinant protein production. Originally, the exclusive function of many of these pathway proteins was believed to be binding at the mitochondria and regulating apoptosis through modulation of the mitochondria permeability. While this protein functionality does still hold true, it is now evident that these proteins also include roles in the metabolic processes of the mitochondria. Furthermore, apoptosis pathway proteins in other organelles within the cell may also both modulate apoptosis and metabolism. This review first details the known links that exist between apoptosis proteins and metabolic functions in the cytosol, mitochondria, and endoplasmic reticulum. Second, the review turns to look at potentially new cell engineering strategies that are linked to metabolism for improving cell culture viability and protein production.     

Cyclophosphamide-induced apoptosis in A431 cells is inhibited by fucosyltransferase IV

Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of difucosylated oligosaccharide LeY which is overexpressed in the cancers derived from the epithelial tissues. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation through the MAPK and PI3K/Akt signaling pathways, but the relationship between FUT4 and apoptosis remained unclear. Here, we investigated the effect of FUT4 overexpression on cyclophosphamide (CPA)-induced apoptosis in A431 cells. Western blot analysis showed that FUT4 overexpression decreased expression of Bax, Caspase 3, and PARP proteins, and increased anti-apoptotic Bcl-2 protein in A431 cells. The anti-apoptosis effect of FUT4 was confirmed both by Annexin-V/PI and JC-1 assays. The results showed that FUT4 overexpression up-regulated phosphorylation of ERK1/2 and Akt which was inhibited by CPA in dose-dependent manner. By blocking the ERK/MAPK and PI3K/Akt pathways with specific inhibitors, we demonstrated that these two pathways were required in mediating the anti-apoptosis effect of FUT4. We concluded that FUT4 inhibited cell apoptosis induced by CPA through decreasing the expression of apoptotic proteins Bax, Caspase 3, and PARP and increasing the expression of anti-apoptotic protein Bcl-2 via the ERK/MAPK and PI3K/Akt signaling pathways in A431 cells.     

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.     

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.     

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm,Bombyx mori

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H₂O₂. In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H₂O₂-induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.     

Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system.     

Deciphering the crucial residues involved in heterodimerization of Bak peptide and anti-apoptotic proteins for apoptosis

B-cell lymphoma 2 (Bcl-2) family proteins are the central regulators of apoptosis, functioning via mitochondrial outer membrane permeabilization. The family members are involved in several stages of apoptosis regulation. The overexpression of the anti-apoptotic proteins leads to several cancer pathological conditions. This overexpression is modulated or inhibited by heterodimerization of pro-apoptotic BH3 domain or BH3-only peptides to the hydrophobic groove present at the surface of anti-apoptotic proteins. Additionally, the heterodimerization displayed differences in binding affinity profile among the pro-apoptotic peptides binding to anti-apoptotic proteins. In light of discovering the novel peptide/drug molecules that contain the potential to inhibit specific anti-apoptotic protein, it is necessary to understand the molecular basis of recognition between the protein and its binding partner (peptide or ligand) along with its binding energies. Therefore, the present work focused on deciphering the molecular basis of recognition between pro-apoptotic Bak peptide binding to different anti-apoptotic (Bcl-xL, Bfl-1, Bcl-W, Mcl-1, and Bcl-2) proteins using advanced Molecular Dynamics (MD) approach such as Molecular Mechanics-Generalized Born Solvent Accessible. The results from our investigation revealed that the predicted binding free energies showed excellent correlation with the experimental values (r² = .95). The electrostatic (ΔG_ele) contributions are the major component that drives the interaction between Bak peptides and different anti-apoptotic peptides. Additionally, van der Waals (ΔG_vdw) energies also play an indispensible role in determining the binding free energy. Furthermore, the decomposition analysis highlighted the comprehensive information about the energy contributions of hotspot residues involved in stabilizing the interaction between Bak peptide and different anti-apoptotic proteins.     

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.     

Effect of recombinant human activated protein C on apoptosis-related proteins

The recombinant human activated protein C (rhAPC) has been reported to reduce mortality in patients with severe sepsis. An anti-apoptotic effect of rhAPC in sepsis is known, but the mechanism through which it acts on the apoptotic pathway is still unclear. Therefore, immunopositivity of the apoptosis-related proteins Bcl-2, an anti-apoptotic protein, c-myc, a proliferative protein, p-21 and p-53, two apoptotic proteins, was determined after rhAPC treatment in a mouse sepsis model. Sepsis was induced by Escherichia coli endotoxin injection. Increased neutrophil infiltration and immunoreactivity to p53 and p21 were observed in the group with sepsis and these immunoreactivities were decreased by rhAPC treatment. In the sepstic group; immunopositivity of Bcl-2 and c-myc was mild and moderate, respectively. In conclusion; p21- and p53-mediated apoptosis was increased in the sepsis model, and for the first time it has been shown that rhAPC decreases sepsis-induced apoptosis resulting from increased p21 and p53 proteins.     

Regulation of apoptosis by BH3 domains in a cell-free system

Background: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins.Results: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system.Conclusions: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.     

提高哺乳动物工程细胞抗凋亡能力的基因策略

利用哺乳动物细胞发酵生产重组蛋白药物具有细菌、酵母等表达系统所不具备的显著优势,因此在生物制药工程中的重要性越来越突出。哺乳动物细胞对工业生产环境下的各种应激环境耐受能力差,易发生细胞凋亡,严重阻碍了大规模生产,降低了生产效率。细胞凋亡是细胞必经的生物学过程,随着对凋亡机制的深入了解,发展出各种抗凋亡策略,并有望应用于重构更适合工业生产的工程细胞。常用的抗凋亡策略包括:下调凋亡蛋白、上调抗凋亡蛋白、增强生长因子自表达、减少有毒代谢产物生成等。以上策略虽然能在一定程度上提高细胞的抗凋亡能力,但距离满足生产的工程细胞重构还有距离,围绕提高工程细胞的抗凋亡能力,已发展出"凋亡工程"这一重要的技术领域。     

Baculoviral infection reduces the expression of four allergen proteins of silkworm pupa

Silkworm (Bombyx mori) larvae are widely used to express exogenous proteins. Moreover, some silkworm pupal proteins can be used as drug‐loading materials for selfexpressed oral tolerance drugs. However, several proteins expressed in silkworm pupae cause severe allergic reactions in humans and animals. Interestingly, some baculovirus vectors have been shown to alter the host gene and its expression in insect cells, but this has not been confirmed in silkworm. Here, we analyzed the effects of infection with an empty B. mori baculovirus (BmNPV) vector on silkworm pupal protein expression. Using a proteomics approach, the allergens thiol peroxiredoxin (Jafrac1), 27‐kDa glycoprotein (p27k), arginine kinase, and paramyosin as well as 32 additional differentially expressed proteins were identified. Downregulation of the messenger RNA expression of the four known allergens was observed after BmNPV infection; subsequent changes in protein expression were confirmed by the western blot analysis using polyclonal antibodies prepared with recombinant proteins of the four allergens. Collectively, these data indicate that the four known allergens of silkworm pupae can be reduced by infection ith an empty BmNPV vector to increase the safety of silkworm pupa‐based exogenous protein expression and drug delivery of oral pharmaceuticals. In addition, the four recombinant allergen proteins may contribute to the diagnosis of allergic diseases of silkworm pupa.     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

Effects of silkworm pupa protein hydrolysates on mitochondrial substructure and metabolism in gastric cancer cells

The effects of silkworm pupa protein hydrolysates (SPPHs) on the apoptosis of MGC-803 gastric cancer cells were investigated in this study. The role of mitochondrial-dependent apoptosis in SPPHs-dependent inhibition of MGC-803 cell viability was also explored. SPPHs were found to induce apoptosis in MGC-803 cells with an IC₅₀ of 0.30 mg/ml.A series of changes in cellular organelle structural were observed during MGC-803 cell apoptosis that included mitochondrial swelling, vacuolation and rupture. These changes may ultimately impact on metabolic energy supply in MGC-803 cells. The expression of the pro- and anti-apoptotic proteins Bcl-2 and Bax, and the activation of cytochrome c (Cyt C), Caspase-3 and Caspase-9 were altered following induction of apoptosis by SPPHs in MGC-803 cells. Moreover, the increase in the ratio of Bax to Bcl-2 expression is known to play an important role in the activation of the mitochondrial-dependent apoptotic pathway.