Effect of temperature on recombinant protein expression in Semliki Forest virus infected mammalian cell lines growing in serum- free suspension cultures.

The firefly luciferase gene was introduced into the Semliki Forest virus (SFV) vector and high titer recombinant SFV particles generated. The broad host range of SFV allowed efficient infection and high level expression of four mammalian cell lines growing in serum-free suspension cultures. The incubation temperature had dramatic effects on the level and duration of recombinant protein expression.

Transient gene expression in mammalian cells grown in serum-free suspension culture.

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method).

SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture.

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture.

The mitochondrial proteins of the neuroblastoma cell line IMR-32.

Mitochondrial proteins exert important functions in biological pathways, particularly they are involved in apoptotic processes. We applied proteomics technologies to analyze the mitochondrial proteins of the neuroblastoma cell line IMR-32, which is often used in apoptosis studies. The proteins were analyzed by two-dimensional (2-D) electrophoresis followed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

Safety aspects related to recombinant protein expression from Semliki Forest virus vectors.

Semliki Forest virus vectors (SFV) have been developed for efficient transgene expression to result in high receptor yields(50-200 pmol receptor/mg protein) in a variety of mammalian host cells. Transfer of the SFV technology to mammalian cells growing in suspension cultures has made it feasible to produce hundreds of milligrams of receptor proteins in a short time. Large-scale production, however, raises the questions of the safety of handling virally infected cells for down-stream processing.

Non-GMP plasmid production for transient transfection in bioreactors.

We describe a generic plasmid purification process for producing DNA for larger-scale transient transfection. Data on plasmid quality with regard to residual protein, endotoxin content and presence of different plasmid forms is given. The effects of contaminants and plasmid forms on expression levels of TNFRp55 and SEAP are discussed.

Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections.

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently.

Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin.

A human prothrombin cDNA has been engineered to obtain a cDNA coding for a secreted form of human prethrombin-2. The secreted prethrombin-2 has been produced in a mammalian expression system using DXB11 cells, a mutant strain of CHO cells in which the dihydrofolate reductase gene has been deleted, and an expression vector carrying the dihydrofolate reductase cDNA. Methotrexate-induced gene amplification favored an efficient production of the recombinant protein which accumulated in the culture medium of the DXB11 cells.

The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties.

The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances. Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines. In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells.

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha.

A fragment (residues His1-Val289) of the alpha chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation.

Isolation of a heregulin-like growth factor secreted by estrogen receptor-negative MDA-MB-231 human breast cancer cells that stimulates estrogen receptor-positive cells.

A 52 kDa peptide growth factor secreted by the estrogen receptor (ER)-negative human breast cancer cell line MDA-MB-231 was purified to homogeneity. It induced transient membrane ruffling, lamellipodia formation, cell motility and proliferation exclusively of ER-positive human breast cancer cells. Partial sequencing revealed a high homology to the protein family of heregulins.

The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit.

Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469).

Expression, purification and crystallization of fully active, glycosylated human interleukin-5.

Recombinant human interleukin-5 (hIL-5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno-affinity chromatography and gel filtration. Purified hIL-5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant.

Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1.

Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse myeloma subclone (J558L-CD4) secreting human CD4-immunoglobulin type G1 receptor (CD4-H gamma 1) was employed as a model system for cell suspension culture and expression of chimaeric molecules. Cells were grown up to 3-5 x 10(6) cells/ml in serum-free and protein-reduced DHI medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Pluronic F68.

Growth study of lactate and ammonia double-resistant clones of HL-60 cells.

The possibility that lactate and ammonia accumulation may have less detrimental effect on cell growth than usually admitted is investigated. We report here the isolation of several HL-60 subclones able to proliferate in the presence of 60 mM sodium lactate and 4 mM ammonium chloride, concentrations usually considered to be toxic for cell proliferation. Growth kinetics and final cell densities of these clones in suspension cultures were similar to the HL-60 cell population in control medium as well as in medium containing ammonia and lactate in which control cells were unable to grow.

Recombinant soluble tumor necrosis factor receptor proteins protect mice from lipopolysaccharide-induced lethality.

The in vivo efficacy of human recombinant soluble tumor necrosis factor (TNF) receptor protein to prevent and to treat lipopolysaccharide (LPS)-induced lethal toxicity in D-galactosamine-treated mice was investigated. Chimeric proteins of the receptor extracellular domains fused to the hinge region of human IgG3 were expressed in myeloma cells (rsTNFR-h gamma 3). The fusion proteins had a disulfide-bonded dimeric structure.

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration.

Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells.

The extracellular domain of the mouse interferon gamma receptor comprising amino acids 17-243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%.

Cloning of the DNA-binding subunit of human nuclear factor kappa B: the level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor alpha.

The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor.

Optimization of culture conditions for high cell density proliferation of HL-60 human promyelocytic leukemia cells.

The purpose of the present investigation was to optimize the culture conditions in suspension of the HL-60 cell line for high-density production. The optimized HL medium was a mixture of RPMI-1640, DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL, Pluronic F68, ethanolamine and selenite. Under these conditions, whether serum was added or not, cells grew to up to 8 x 10(6) cells ml-1, which was at least three times higher than the maximum cell density usually described.

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells.

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity.

Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies.

The pleiotropic cyto/lymphokine tumor necrosis factor (TNF) exerts its functions by binding to specific cell-surface receptors. We have prepared two sets of monoclonal antibodies (mAbs) against TNF-binding proteins from the HL-60 (htr-mAb series) and U-937 (utr-mAb series) cell lines. The htr antibodies inhibit the binding of 125I-labeled TNF-alpha to HL-60 cells only partially, whereas they block the TNF-alpha binding to several adenocarcinoma cell lines (HEp-2, HeLa, and MCF7) almost completely.

Proteolytic activity in the culture supernatants of mouse hybridoma cells.

When serum-reduced or serum-free culture supernatants were incubated for 16 hours at 37 degrees C, more than 70-80% of original proteins were digested as measured by gel electrophoretic analysis. The proteolytic activity, which was only observed at pH values lower than 4.5, was reduced in conditioned medium containing higher concentrations of fetal calf serum.