High Levels Of Recombinant Proteins Research Articles

DRB4-Dependent TAS3 trans-Acting siRNAs Control Leaf Morphology through AGO7

trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.

479. Development and Characterization of a New Noncytopathic Vector Derived from Semliki Forest Virus

Top of pageAbstract Alphavirus vectors can express high levels of recombinant proteins in many different cell types. However, this expression is transient, due to the cytopathic nature of viral replication. Several noncytopathic mutants have been obtained from Sindbis virus (SIN) and Semliki Forest virus (SFV), which could be used when sustained transgene expression is desired. However, these noncytopathic vectors are generally only able to replicate in a limited number of cell lines, and have a low packaging effciency. In order generate new SFV noncytopathic vectors we have introduced mutations described to turn SIN noncytopathic into conserved positions in SFV. Interestingly, we found that one of the vectors generated in this way, which contained mutation P718T in nsp2, while apparently not able to replicate in most cells, gave rise with a relative high frequency to noncytopathic variants that formed colonies without selection when using LacZ as a reporter gene. We rationalized that these noncytopathic variants were due to second mutations that appeared naturally in this vector. To isolate these noncytopathic variants we inserted the pac gene into the SFV P718T mutant and puromycine resistant cell clones were selected in BHK cells. The noncytopathic replicons were then cloned by RT-PCR. As hypothesized, one noncytopathic replicon was rescued in this way containing in addition to P718T a second mutation in the nuclear localization signal of nsp2. This noncytopathic mutant had a lower level of replication as compared to the wild type replicon, but it was able to express heterologous proteins at similar levels. Further characterization of this double mutant will be discussed in detail.

1P140 A novel expression system for high-level recombinant protein production in Escherichia coli(4. Protein engineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P140 A novel expression system for high-level recombinant protein production in Escherichia coli(4. Protein engineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.

Aqueous extraction of maize endosperm: Insights for recombinant protein hosts based on downstream processing

The use of transgenic plants for the production of heterologous proteins for pharmaceutical applications has increased continuously, since this provides a viable alternative to traditional expression systems. The choice of the host crop for such recombinant proteins has a significant influence on downstream processing and overall process economics. Varieties with less oil and phenolic compounds generate a less complex extract, thus facilitating recovery and purification processes for the heterologous protein. Maize is a crop which has proved its potential for the production of high levels of recombinant proteins. In this study, the aqueous extracts of the endosperm of six non-transgenic maize varieties were analyzed for evaluation of their potential as hosts for the production of recombinant proteins. This evaluation was based on the following parameters associated with downstream processing: extract filtrability, concentration of proteins, carbohydrates and phenolics compounds and flour oil content. The study of the extraction process was carried out using factorial design and response surface methodology. The argument for the selection of a specific maize variety is presented as well as a discussion of the optimum range of conditions for the minimization of compounds, which could negatively interfere with downstream processing operations.

High-level recombinant protein production by overexpression of Mlc in Escherichia coli

Escherichia coli excretes acetate during aerobic growth on LB broth containing glucose and growth ceases before depletion of glucose because of the low pH caused by the accumulation of acetate. It has been known that the acetate accumulation is reduced even when E. coli is grown in the presence of high concentration of glucose if Mlc is overexpressed. The intracellular concentration of Mlc is very low in E. coli because of autoregulation and a low efficiency of mlc translation. We constructed various mutants that can express higher levels of Mlc using site-directed mutagenesis and one of the Mlc-overproducing mutant showed reduced glucose consumption rate and low production of acetate. The mutant showed higher foreign gene expression level than that of its parental strain in the presence of glucose. These results suggest that the Mlc overproducing E. coli strain having an improved ability of glucose utilization can be a better host for high-level production of useful recombinant proteins.

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Optimisation and evaluation of a high-throughput mammalian protein expression system

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Aqueous Extraction of Recombinant Human Proinsulin from Transgenic Maize Endosperm

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.

High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification

We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector.

Serum-free large-scale transient transfection of CHO cells.

To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 x 10(6) cells/ml were transfected with 2.5 microg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10

An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.

Generation of high-recombinant- protein-producing Chinese hamster ovary (CHO) cells.

The purpose of this chapter is to propose a practical procedure for the generation and selection of Chinese hamster ovary cells producing high levels of recombinant protein by combining in vitro and in vivo amplification of the foreign gene. A detailed description of the expression and amplification plasmids utilized for the in vitro generation of long DNA concatenamers, as well as the cell transfection and selection protocols are given. The procedure required for in vivo gene amplification using the dihydrofolate reductase/methotrexate system is also described.

Isolation of a novel Thermus thermophilus metal efflux protein that improves Escherichia coli growth under stress conditions.

The mechanisms of metal ion transport in thermophilic organisms are poorly understood. Phage display-based screening of a Thermus thermophilus genomic library in Escherichia coli led to the identification of a novel metal cation efflux protein. The Thermus protein showed extensive sequence and putative structural conservation to Czr and Czc proteins in mesophilic bacterial and mammalian species. Expression of the gene in E. coli led to increased resistance to zinc and cadmium ions, but not to cobalt, in an effect that was apparently caused by increased efflux of metals from the cell. This increased resistance was inducible by zinc and cadmium and, to a lesser extent, by cobalt. Furthermore, E. coli cells containing the Thermus gene exhibited improved cell physiology and delayed cell lysis during recombinant protein production, leading to accumulation of higher levels of recombinant protein. The molecular basis and potential application of the findings are discussed.

Heterologous expression, purification, and immunological reactivity of a recombinant HSP60 from Paracoccidioides brasiliensis.

The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

Cloning of the bovine immunodeficiency virus gag gene and development of a recombinant-protein-based enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

Recombinant Semliki forest virus infects and kills human prostate cancer cell lines and prostatic duct epithelial cells ex vivo.

Semliki Forest Virus (SFV) is a broad host range RNA virus capable of high-level recombinant protein expression and apoptosis induction in many cell types. We have successfully used a recombinant, replication deficient SFV vector to express the LacZ marker gene product in seven human prostate cell lines, as well as in human prostate tissue explants. Flow cytometry revealed that 40-60% of PPC-1 prostate cancer cells died 24-72 h after infection with SFV-LacZ virus. Most human prostate cancer cell lines expressed high levels of recombinant protein. Infection of human prostate tissue ex vivo led to similarly high expression levels but the recombinant beta-galactosidase was confined to duct epithelial cells. Infection of cell and tissue cultures resulted in detachment of adherent cells from the culture surface and detachment of epithelial cells from the basement membrane of tissue. Our results indicate that SFV may be useful in targeting recombinant protein expression and apoptosis to prostatic duct epithelial cells.

Subcellular targeting of multiligand-binding protein gC1qR

gC1q receptor, a protein originally described as the cell surface receptor for the globular heads of complement factor C1q, has been found to bind human H-kininogen with high affinity and specificity. Therefore, gC1qR has been considered candidate kininogen docking site on the surfaces of platelets, neutrophils and endothelial cells. Recent work demonstrating that gC1qR is an intracellular protein that is tightly associated with mitochondria rather than targeted to the cell surface has challenged this view. To further probe cellular trafficking routes of gC1qR, we overexpressed human gC1qR in a mammalian cell and monitored cell surface exposure of recombinant gC1qR by virtue of its capacity to bind labeled H-kininogen. Transient transfection of COS1 cells with the full-length cDNA of human gC1qR resulted in a high level of recombinant protein that matched the pool of endogenous gC1qR present in these cells. Overexpression of gC1qR did not significantly increase the number of H-kininogen binding sites exposed by the transfected cells thus denying the possibility that alternative routing of gC1qR to the surface of COS1 cells occurs at significant levels. Hence gC1qR has the capacity to tightly bind H-kininogen, but because gC1qR is routed to mitochondria it cannot fulfill the postulated functions as a cell docking site for kininogens and complement factors.

Transgenic plants expressing human glutamic acid decarboxylase (GAD65), a major autoantigen in insulin-dependent diabetes mellitus

Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65.

Development of a Bicistronic Vector Driven by the Human Polypeptide Chain Elongation Factor 1α Promoter for Creation of Stable Mammalian Cell Lines That Express Very High Levels of Recombinant Proteins

We have developed a new vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pEFIRES-P, both the recombinant cDNA and the puromycin resistant gene (pac) are transcribed as a single message driven by the strong human polypeptide chain elongation factor 1α promoter. The presence of an internal ribosome entry site ensures any clones that are resistant to puromycin also express high levels of recombinant protein encoded by cDNA cloned upstream from thepacgene. We report the use of this vector to generate stable cell lines expressing human pyruvate carboxylase and show that following transfection and clonal selection, all twenty colonies that conferred high dose resistance to puromycin also expressed very high levels of functional pyruvate carboxylase. Use of pEFIRES-P should permit the rapid and efficient generation of stable cell lines for the characterisation of recombinant proteins.