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.     

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

B型流感病毒血凝素的表达及免疫原性测定北大核心CSCD

B型流感病毒是引起季节性流感的原因之一,严重时会造成重大疾病或死亡。为了检测B型流感病毒2个疫苗候选毒株的血凝素(hemagglutinin,HA)蛋白胞外段在哺乳动物细胞中的表达及在小鼠体内的免疫原性,本研究将带有三聚体标签的HA胞外段(HA-ectodomain,HA-ecto)序列及神经氨酸酶(neuraminidase,NA)全长编码框经密码子优化后构建至pCAGGS载体中,通过线性聚乙烯亚胺将pCAGGS-HA-ecto与pCAGGS-NA共转染293T细胞。收集转染后96h的上清,通过镍离子亲和层析及分子筛层析获得三聚体形式的HA-ecto蛋白,然后将HA-ecto三聚体蛋白免疫小鼠,进行酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)及血凝抑制实验(hemagglutination inhibition,HAI)检测HA-ecto蛋白诱导小鼠后产生的抗体水平。纯化结果显示,通过哺乳动物细胞表达系统能够得到分泌型表达的三聚体HA-ecto蛋白。ELISA及HAI结果显示,三聚体HA-ecto蛋白二次免疫小鼠后,能诱导小鼠产生较高水平的同源和异源交叉抗体。以上结果表明,哺乳动物细胞表达的B型流感病毒HA蛋白可作为亚单位重组流感疫苗的候选。     

Relationships between hepatitis C virus replication and CXCL-8 production in vitro

The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.     

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins

Background  

Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.     

Different Immunity Elicited by Recombinant H5N1 Hemagglutinin Proteins Containing Pauci-Mannose,High-Mannose,or Complex Type N-Glycans

Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA), a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9) cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic) and mammalian cells (CHO). While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.     

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.     

Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method

We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.     

Functional expression of mammalian receptors and membrane channels in different cells

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Codon engineering for improved antibody expression in mammalian cells

While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.     

Accelerated cell line development using two-color fluorescence activated cell sorting to select highly expressing antibody-producing clones

The success of engineered monoclonal antibodies as biopharmaceuticals has generated considerable interest in strategies designed to accelerate development of antibody expressing cell lines. Stable mammalian cell lines that express therapeutic antibodies at high levels typically take 6-12 months to develop. Here we describe a novel method to accelerate selection of cells expressing recombinant proteins (e.g., antibodies) using multiparameter fluorescence activated cell sorting (FACS) in association with dual intracellular autofluorescent reporter proteins. The method is co-factor-independent and does not require complex sample preparation. Chinese hamster ovary (CHO) clones expressing high levels of recombinant antibody were selected on the basis of a two-color FACS sorting strategy using heavy and light chain-specific fluorescent reporter proteins. We were able to establish within 12 weeks of transfection cell lines with greater than a 38-fold increase in antibody production when compared to the pool from which they were isolated, following a single round of FACS. The method provides a robust strategy to accelerate selection and characterization of clones and builds a foundation for a predictive model of specific productivity based upon on two-color fluorescence.     

Cell-specific effects of human cytomegalovirus unique region on recombinant protein expression

The major immediate-early (MIE) promoter of human cytomegalovirus (CMV) is widely used to express recombinant proteins in mammalian cells. CMV MIE promoter contains a strong enhancer and an AT-rich unique region (UR). The UR can function as an insulator or a negative element of CMV MIE promoter, depending on the cellular proteins associated with it. To examine the effects of UR on recombinant protein expression in mammalian cells, we constructed two CMV MIE promoter-based expression plasmids for comparison to the conventional CMV MIE promoter by removing or adding UR. Addition of UR enhances transgene expression in HEK293 stable cells while removal of UR increases both transient and stably integrated expression in HeLa cells. Our results further demonstrate that the cell-specific effect of UR depends on the protein levels of UR-binding proteins, pancreatic-duodenal homeobox factor-1, special AT-rich sequence binding protein 1, and CCAAT displacement protein, in these cells. Collectively, these modified CMV expression plasmids can be utilized to improve recombinant protein production in specific mammalian cell lines.     

Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.     

A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.     

Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon-induced Mx proteins.

In mouse Mx+ cells, interferon alpha/beta induces the synthesis of the nuclear Mx protein, whose accumulation is correlated with specific inhibition of influenza viral protein synthesis. When Mx+ mouse cells are microinjected with the monoclonal anti-Mx antibody 2C12, interferon alpha/beta still induces Mx protein, but no longer inhibits efficiently the expression of influenza viral proteins as visualized by immunofluorescent labeling. However, interferon inhibition of an unrelated control virus, vesicular stomatitis virus, remains unchanged. Proteins with homology to mouse Mx protein are found in interferon-treated cells of a variety of mammalian species. In rat cells, for instance, rat interferon alpha/beta induces three Mx proteins which all cross-react with antibody 2C12 but differ in mol. wt and intracellular location, and it protects these cells well against influenza viruses. However, when rat cells are microinjected with antibody 2C12, interferon alpha/beta cannot induce an efficient antiviral state against influenza virus infection, whereas protection against vesicular stomatitis virus is not altered. These results show that both mouse and rat cells require functional Mx proteins for efficient protection against influenza virus. They further demonstrate that microinjection of antibodies is a promising way of elucidating the role of particular interferon-induced proteins in the intact cell.     

Cell‐free production of a therapeutic protein: Expression,purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell‐free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell‐free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell‐free system using instrumented mini‐bioreactors for highly reproducible protein production. We achieved recombinant protein production (～600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell‐free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag‐free self‐cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose‐level production of therapeutic proteins at the point‐of‐care.     

Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus

Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer; the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind alpha2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as individual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.