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1.
Protein expression and secretion in insect cells have been widely studied in the baculovirus-infected insect cell system. In directly transfected insect cells only intracellular expression and purification of recombinant proteins have been studied in detail. To examine multiple recombinant protein variants, easy and fast expression and a purification screening system are required. The aim of this study was to establish an effective and rapid secretion system for human azurocidin using directly transfected insect cells. We also constructed and tested expression vectors possessing heterologous signal peptides derived from human azurocidin, yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1), and papaya papain IV to secrete yellow lupin and red kidney bean purple acid phosphatases, PPD1, and papain IV. Our results demonstrate that the secretion vectors used here can direct recombinant proteins to the culture medium very effectively, allowing their simple purification on a small/medium scale. Based on secretion and activity analyses it seems that the azurocidin signal peptide is one of the most potent secretion signals.  相似文献   

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重组抗体药物在抗肿瘤、抗自身免疫病、抗感染等领域都有较好的应用前景,且市场的需求量正在逐年上升。然而许多的抗体药物在研究、生产过程中都面临着产量低、稳定性差、活性低等问题。目前,在大规模制备抗体药物的过程中,常常利用动物细胞表达系统来生产分泌蛋白,而信号肽作为连接在分泌蛋白N端的一段氨基酸序列,能够控制蛋白质的分泌途径进而影响抗体蛋白的表达产量。简要概述了信号肽的一级结构与作用机制,同时,指出其对重组抗体蛋白分泌效率的影响途径,最后结合各种研究成果总结出信号肽序列优化策略。  相似文献   

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Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.  相似文献   

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Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.  相似文献   

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Most biotherapeutic drugs are recombinant monoclonal antibodies which are mostly produced in monoclonal cell lines derived from Chinese hamster ovary (CHO) cells. Various clones expressing a monoclonal recombinant antibody were analyzed and a correlation of the antibody concentration and the relative mRNA level of calreticulin (CALR), glucose‐regulated protein 78 and 94 kDa (GRP78, GRP94) and spliced X‐box binding protein 1 (XPB1) was observed. By means of these results we were motivated to establish a novel selection system based on endoplasmic reticulum (ER) stress, which allows the rapid identification and isolation of high‐expressing clones out of a pool mainly consisting of low‐ and medium‐producing cells. Several ER stress responsive elements were tested with the aid of a recombinase mediated cassette exchange (RMCE) procedure. Very surprisingly, only GRP78 reporter constructs were strongly stimulated upon antibody expression. Furthermore we found that GRP78 reporter constructs are very suitable to reflect the level of antibody expression (IgG) in recombinant CHO cells. Based on these results, it is concluded, that the novel ER stress based selection system developed during this study is suitable to identify and isolate clones with a high level of antibody expression. Biotechnol. Bioeng. 2012; 109: 2599–2611. © 2012 Wiley Periodicals, Inc.  相似文献   

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An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high‐yielding Chinese hamster ovary (CHO) transient expression system. Suspension‐adapted CHO‐K1 host cells were engineered to express the gene encoding Epstein‐Barr virus (EBV) nuclear antigen‐1 (EBNA‐1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA‐1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA‐1‐only transfectants. This was confirmed with the retransfection of an EBNA‐1‐only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA‐1‐only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:132–141, 2014  相似文献   

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We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (KA = 1.9 × 105?1.4 × 108 M?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 × 105?1.8 × 107 M?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.  相似文献   

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Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4+ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence.  相似文献   

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IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native‐like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or “hetero‐mAbs” using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469–477, 2017  相似文献   

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Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MSn experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified.  相似文献   

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IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8?/? cell lines was fourfold higher than in the best‐producing clones of FUT8?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.  相似文献   

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We have undertaken the production of recombinant soluble Fc epsilon receptor II (Fc epsilon RII) as a secretory protein, but not as a cleavage product of membrane-bound receptor. Several plasmid constructs containing soluble receptor sequence were prepared. Only a chimeric gene containing the sequences encoding IL-6 signal peptide and the soluble moiety of Fc epsilon RII could be expressed in Xenopus laevis oocytes and CHO cells, resulting in the secretion of soluble Fc epsilon RII. The recombinant soluble Fc epsilon RII was also produced in the yeast expression system. The NH2-terminal sequence analysis of the chimeric gene product generated by oocytes demonstrated the correct cleavage of IL-6 leader sequence by a signal peptidase. Moreover, most of CHO cell and all of the yeast-derived recombinant molecules were products identical with the native B cell-derived soluble Fc epsilon RII. These recombinant products as well as the natural soluble receptor derived from a human B cell line could bind both human IgE and two different anti-Fc epsilon RII mAb and could competitively inhibit the binding of IgE to Fc epsilon RII-expressing cells. However, the recombinant soluble Fc epsilon RII and highly purified native molecules did not display any B cell growth-promoting activity.  相似文献   

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hK-Fc融合蛋白的改良、表达及其生物活性的分析   总被引:1,自引:0,他引:1  
为了延长人激肽释放酶(hK)的血清半衰期,提高分泌蛋白的产率,制备了重组激肽释放酶-IgG1 Fc融合蛋白(hK'-Fc)。采用PCR扩增hK基因和IgG1的Fc序列,用鼠源信号肽序列替换hK基因原有的信号肽序列,构建改良型融合蛋白hK'-Fc以及天然型融合蛋白hK-Fc的表达载体,转染中国仓鼠卵巢细胞(CHO)细胞,筛选稳定分泌融合蛋白的细胞株,通过Western blotting鉴定信号肽改造效果,利用Protein A+G亲合层析柱纯化融合蛋白,酶学实验检测融合蛋白的体外活性。结果表明:成功构建了pcDNA-hK'-Fc以及pcDNA-hK-Fc重组表达载体;获得了稳定表达融合蛋白的细胞株,产量达11mg/L以上;信号肽改造后融合蛋白的分泌效率提高约5~10倍;融合蛋白能水解其特异性的底物S-2266,具有生物学活性。本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础,也为研制治疗脑梗塞疗效更好的第二代hK蛋白和其他药用蛋白的改良提供新的线索。  相似文献   

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Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR‐deficient DG44, and DUXB11‐based DHFR deficient CHO. Current Genentech commercial full‐length antibody products have all been produced in the DUXB11‐derived DHFR‐deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11‐derived DHFR‐deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11‐based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14‐day fed batch cultures in shake flasks. In contrast, the DUXB11‐based host produced ~0.1 g/l for both antibodies in the same 14‐day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ~2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:980–985, 2013  相似文献   

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Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal‐derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum‐free, protein‐free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single‐cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD‐CHO? and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012  相似文献   

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As the main immunogen that could stimulate neutralized antibody in pigs, recombinant E2 protein of CSFV was expressed in CHO‐dhfr?cells driven by endogenous Txnip promoter from Chinese hamster. Different fragments of Txnip promoter were amplified by PCR from isolated genomic DNA of CHO cells and cloned into different expression vectors. Compared with CMV promoter, CHO‐pTxnip‐4‐rE2 (F12) cell clone with the highest yield of rE2 protein was established by random insertion of the expression cassette driven by 860 bp sequences of Txnip promoter. In combination with treatment of 800 nM MTX for copy amplification of inserted expression cassette, the dynamic expression profile of rE2 protein was observed. Then inducible expression strategy of balance between viable cell density and product yield was conducted by mixed addition of 0.1 mM NADH and 0.1 mM ATP in culture medium at day 3 of batch‐wise culture. It could be concluded that Txnip promoter would be a promising alternative promoter for recombinant antigen protein expression in transgenic cells.  相似文献   

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