外源蛋白在中国仓鼠卵巢细胞中高效表达的策略

高效表达外源蛋白,在生物制药中有重要意义.中国仓鼠卵巢细胞（Chinese hamster ovary cell）是表达外源蛋白的最佳真核表达系统之一.影响外源蛋白在CHO细胞表达的因素甚多,主要包括载体、宿主细胞和外源基因几方面.深入了解和灵活运用它们之间的关系,有助于获得外源基因在CHO细胞中的高效表达.     

CHO细胞表达系统启动子

中国仓鼠卵巢（Chinese hamsters ovary,CHO）细胞是目前重组蛋白质生产的首选宿主细胞。利用CHO细胞生产重组蛋白质,启动子是启动转基因转录的关键。核心启动子是RNA聚合酶与转录起始复合物集合的部位,分为集中型和分散型两种类型。目前,CHO细胞常用的启动子为病毒启动子、异源启动子、内源性和诱导性启动子等。也可以利用合成生物学及相关的数据库,人工设计合成启动子及鉴定新型启动子。本文综述了CHO细胞常用的启动子以及人工设计的合成启动子在CHO细胞中重组蛋白质表达方面的进展,为哺乳动物细胞选择合适的启动子,保证蛋白质表达量最大化,并确保长时间表达稳定性提供参考。     

CHO细胞表达系统启动子

中国仓鼠卵巢（Chinese hamsters ovary,CHO）细胞是目前重组蛋白质生产的首选宿主细胞。利用CHO细胞生产重组蛋白质,启动子是启动转基因转录的关键。核心启动子是RNA聚合酶与转录起始复合物集合的部位,分为集中型和分散型两种类型。目前,CHO细胞常用的启动子为病毒启动子、异源启动子、内源性和诱导性启动子等。也可以利用合成生物学及相关的数据库,人工设计合成启动子及鉴定新型启动子。本文综述了CHO细胞常用的启动子以及人工设计的合成启动子在CHO细胞中重组蛋白质表达方面的进展,为哺乳动物细胞选择合适的启动子,保证蛋白质表达量最大化,并确保长时间表达稳定性提供参考。     

Effect of PDI overexpression on recombinant protein secretion in CHO cells

In eukaryotic cells, protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. Increasing PDI activity in bacterial, yeast, and insect cell expression systems can lead to increased secretion of heterologous proteins containing disulfide bridges. Since Chinese hamster ovary (CHO) cells are widely used for the expression of recombinant proteins, we expressed recombinant human PDI (rhu PDI) in CHO cells to increase cellular PDI levels and examined its effect on the secretion of two different recombinant proteins: interleukin 15 (IL-15) and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). Secretion of TNFR:Fc (a disulfide-rich protein) is decreased in cells overexpressing PDI; the TNFR:Fc protein is retained inside these cells and colocalizes with the overexpressed rhu PDI protein in the endoplasmic reticulum. PDI overexpression did not result in intracellular retention of IL15. The nature of the interaction between PDI and TNFR:Fc was further investigated by expressing a disulfide isomerase mutant PDI in CHO cells to determine if the functional activity of PDI is involved in the cellular retention of TNFR:Fc protein.     

重组蛋白在中国仓鼠卵巢细胞中高效表达的影响因素

高效表达重组蛋白 ,对于生物制药意义重大。大多数药用蛋白是糖蛋白 ,中国仓鼠卵巢细胞 (Chinesehamsterovarycell,CHO)是目前重组糖基蛋白生产的首选体系。影响外源蛋白在CHO细胞中表达的因素很多 ,从CHO细胞表达体系、表达载体系统、外源基因、表达细胞株的加压扩增与筛选、细胞大规模培养等方面对CHO高效表达加以阐述 ,同时提出存在的问题和未来的发展方向。     

RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells

RNA interference (RNAi) technology has become a novel tool for silencing gene expression in cells or organisms, and has also been used to develop new therapeutics for certain diseases. This review describes its other application of using RNAi technology to increase cellular productivity and the quality of recombinant proteins that are produced in Chinese hamster ovary (CHO) cells, the most important mammalian cell line used in producing licensed biopharmaceuticals in these days. The approaches reported include the silencing of apoptosis-associated gene expression, protein glycosylation-associated gene expression, lactate dehydrogenase involved in cellular metabolism, and dihydrofolate reductase used for gene amplification. All of these works belong to the single component approach therefore depends strongly on the identification of the down-regulation of the critical target gene which can markedly influence the cellular functions associated with recombinant protein expression in CHO cells. Future RNAi approaches can be extended to silence multiple targets involved in different cellular pathways for changing the global gene regulation in cells, as well as the targets related to microRNA molecules for cellular self regulation.     

Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.     

Post‐translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37°C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32°C but was diminished at lower temperatures. The expression of both cap‐dependent and cap‐independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32°C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post‐translational events appear to proceed with higher fidelity and accuracy at 32 than 37°C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32°C recombinant cap‐dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post‐translational processing. Biotechnol. Bioeng. 2010;105: 215–220. © 2009 Wiley Periodicals, Inc.     

Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels

In order to avoid the metabolic burden of protein expression during cell growth, and to avoid potential toxicity of recombinant proteins, microbial expression systems typically utilize regulated expression vectors. In contrast, constitutive expression vectors have usually been utilized for isolation of protein expressing mammalian cell lines. In mammalian systems, inducible expression vectors are typically utilized for only those proteins that are toxic when overexpressed. We developed a tetracycline regulated expression system in CHO cells, and show that cell pools selected in the uninduced state recover faster than those selected in the induced state even though the proteins showed no apparent toxicity or expression instability. Furthermore, cell pools selected in the uninduced state had higher expression levels when protein expression was turned on only in production cultures compared to pools that were selected and maintained in the induced state through production. We show a titer improvement of greater than twofold for an Fc-fusion protein and greater than 50% improvement for a recombinant antibody. The improvement is primarily due to an increase in specific productivity. Recombinant protein mRNA levels correlate strongly with protein expression levels and are highest in those cultures selected in the uninduced state and only induced during production. These data are consistent with a model where CHO cell lines with constitutive expression select for subclones with lower expression levels.     

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells

Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co‐expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over‐expression of the yeast ‘protein disaggregase’ Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over‐expression of the yeast or mammalian Hsp70/40 chaperones. Over‐expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5‐fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over‐expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over‐expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells. Biotechnol. Bioeng. 2010; 105: 556–566. © 2009 Wiley Periodicals, Inc.     

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.     

Change of insulin-like growth factor gene expression in Chinese hamster ovary cells cultured in serum-free media

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium. When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements-including insulin, transferrin, ethanolamine, and selenium-were removed from MED-3, theIGF expression was consistently down-regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level ofIGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.     

Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post-translational processing of the mature rhBMP-2 dimer. PACEsol co-expression was also associated with a significant increase (almost four-fold) in cellular productivity of rhBMP-2 protein. Differential proteomic expression profiling using 2-D DIGE and MALDI-TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.