Demonstration of a second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into fluorescent liposomes.

Recombinant full-length human CD23 has been incorporated into fluorescent liposomes to demonstrate the existence of a ligand for CD23 that is different from the previously known ligand, immunoglobulin E (IgE). The novel ligand for CD23 is expressed on subsets of normal T cells and B cells as well as on some myeloma cell lines. The interaction of full-length CD23 with its ligand is specifically inhibited by anti-CD23 monoclonal antibodies and by IgE, and it is Ca2+ dependent. Moreover, tunicamycin treatment of a CD23-binding cell line, RPMI 8226, significantly reduced the binding of CD23 incorporated into fluorescent liposomes, and a sugar, fucose-1-phosphate, was found to inhibit CD23-liposome binding to RPMI 8226 cells, suggesting the contribution of sugar structures on the CD23 ligand. In addition, CD23-transfected COS cells were shown to form specific conjugates with the cell line RPMI 8226. These data demonstrate that CD23 interacts with a ligand, which is different from IgE, and that CD23 can be considered as a new surface adhesion molecule involved in cell-cell interactions.

Use of glucose consumption rate (GCR) as a tool to monitor and control animal cell production processes in packed-bed bioreactors.

For animal cell cultures growing in packed-bed bioreactors where cell number cannot be determined directly, there is a clear need to use indirect methods that are not based on cell counts in order to monitor and control the process. One option is to use the glucose consumption rate (GCR) of the culture as an indirect measure to monitor the process in bioreactors. This study was done on a packed-bed bioreactor process using recombinant CHO cells cultured on Fibra-Cel disk carriers in perfusion mode at high cell densities. A key step in the process is the switch of the process from the cell growth phase to the production phase triggered by a reduction of the temperature. In this system, we have used a GCR value of 300 g of glucose per kilogram of disks per day as a criterion for the switch. This paper will present results obtained in routine operations for the monitoring and control of an industrial process at pilot-scale. The process operated with this GCR-based strategy yielded consistent, reproducible process performance across numerous bioreactor runs performed on multiple production sites.

Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks.

Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.

Repeated hybridoma batch culture with cell recycle.

At the end of a hybridoma batch culture, the cells are usually discarded after separation from the culture broth. If, however, they are aseptically recycled into the reactor, the production process can be resumed simply by the addition of fresh medium. This cycle can then be repeated several times consecutively. In a test case, with a mouse hybridoma, we found antibody yields for each cycle in the same range as for a standard batch. In a 15 1 stirred tank reactor we could, within 6 days, produce 2.8 g of monoclonal antibody (MAb). This type of reactor operation allowed a doubling in the reactor volumetric productivity (mg/l/day).

Recombinant protein production using the Semliki Forest Virus expression system.

We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial ß-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 10(9) cells(-1)) with an average specific activity of 3942 ± 765 pg PGE(2) µg(-1) microsomal protein 5 min(-1).

Large scale transient expression with COS cells.

We demonstrate here that transient expression with COS cells can be performed at the one litre scale for a period of more than 10 days. Cells grown in T225 flasks were transfected by electroporation, transferred into spinners, and then grown either in suspension or on microcarriers. A daily medium change significantly extented culture life and production time, compared with standard protocols.Concentrations of the product, the secreted fusion protein CD40-Fc, were comparable in microcarrier and suspension culture. Cultures were started in fetal calf serum containing medium and the subsequent production process was performed in a low protein serum free medium which allowed easy downstream processing. 10 litres of supernatant, collected from one transfected batch of cells, yielded 30 mg of purified and biologically active protein.In addition to developing a simplified protocol for generation of cells we also reduced the material (DNA, cuvettes) required for electroporation. Our results show that scale up of transient expression to the litre scale can be successfully acieved. This provides a new tool to generate milligram quantities of protein within weeks of gene cloning.

In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase.

Phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. The enzyme is a metalloenzyme which contains 1 mol of zinc per mol of enzyme. Heterologous expression of the cDNA coding for the Candida albicans enzyme in the prokaryotic host Escherichia coli results in an expression level of up to 30% of total E. coli protein. Ten percent of recombinant PMI is expressed in the soluble fraction and 90% in inclusion bodies. Inclusion of a high level of zinc in the fermentation medium resulted in a fourfold increase in soluble protein. Co-expression of the bacterial chaperones, GroES and GroEL, resulted in a proportional twofold increase in soluble PMI while causing an overall decrease in the PMI expression level. Folding denatured PMI in vitro required reductant and zinc ions. The yield of renatured protein was increased by folding in the presence of GroEL and DnaK in an ATP-independent manner. The refolding yield of denatured soluble enzyme from a guanidine solution was threefold higher than that of folding monomerized inclusion body protein solubilized in guanidine hydrochloride. This suggests that a proportion of recombinant protein expressed in E.coli inclusion bodies may be irreversibly denatured.

Expression of ligand-gated ion channels with the Semliki Forest virus expression system.

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system. The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P2x virus stocks. Infection of BHK, CHO and RIN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 x 10(6) 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT3His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 x 10(6) receptors per cell.

Selenomethionine labelling of phosphomannose isomerase changes its kinetic properties.

Phosphomannose isomerase (PMI) is an essential enzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the three-dimensional structure of the enzyme from the pathogen Candida albicans, we have produced the recombinant selenomethionine-labelled enzyme (SeMet-PMI). DL41, a methionine auxotroph Escherichia coli strain, was transformed with a PMI expression plasmid and grown on an enriched selenomethionine-containing medium to high-cell densities. The SeMet-PMI protein has been purified and found by amino acid analysis to have its methionine residues replaced by selenomethionine residues. Electrospray mass spectroscopy showed a major species of 49,063 +/- 10 Da for SeMet-PMI compared to 48,735 +/- 6 Da for the normal recombinant enzyme, accounting for the incorporation of seven selenomethionine residues. SeMet-PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet-PMI showed that its Km for the substrate mannose-6-phosphate was fourfold higher than that of its methionine-containing counterpart. The inhibition constant for zinc ions was also increased by a similar factor. However, the Vmax was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate-binding pocket in the active site, rendering substrate access more difficult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the active site.

A bacterial signal peptide directs efficient secretion of eukaryotic proteins in the baculovirus expression system.

Escherichia coli remains an organism of choice for the production of recombinant proteins required in large quantities. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. Our efforts to use E. coli to secrete a human CD23 soluble variant fused to a pair of IgG binding domains via the Staphylococcal protein A signal peptide were unsuccessful. Surprisingly, when the same construct was expressed in the baculovirus system, efficient secretion was observed and cleavage of the signal peptide occurred at the expected site. Varying the genes in the fusions or the tags, or the topology of the gene and the tag, did not affect the high-level secretion and cleavage at the correct site. We envision that fusion of the bacterial signal sequence to eukaryotic recombinant genes will prove to be a tool of value for efficient protein secretion in insect cells using the baculovirus expression system.

The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products.

The baculovirus expression vector system was employed to produce human apolipoprotein E and ß-galactosidase in order to study the effect of multiplicity of infection on secreted and non-secreted recombinant protein production. Prior knowledge of the influence of other cell culture and infection parameters, such as the cell density at time of infection and the time of harvest, allowed determination of the direct and indirect influences of multiplicity of infection on recombinant protein synthesis and degradation in insect cells. Under non-limited, controlled conditions, the direct effect of multiplicity of infection (10(-1)-10 pfu/cell) on specific recombinant product yields of non-secreted ß-galactosidase was found to be insignificant. Instead, the observed increased in accumulated product was directly correlated to the total number of infected cells during the production period and therefore ultimately dependent on an adequate supply of nutrients. Only the timing of recombinant virus and protein production was influenced by, and dependent on the multiplicity of infection. Evidence is presented in this study that indicates the extremely limited predictability of post-infection cell growth at very low multiplicities of infection of less than 0.1 pfu/cell. Due to the inaccuracy of the current virus quantification techniques, combined with the sensitivity of post-infection cell growth at low MOI, the possibility of excessive post-infection cell growth and subsequent nutrient limitation was found to be significantly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination at low multiplicity of infection is investigated for a secreted recombinant form of human apolipoprotein E. Comparison of human apolipoprotein E production and secretion at multiplicities of infection of 10(-4)-10 pfu/cell revealed increased product degradation and contamination with intracellular proteins at low multiplicities of infection.

Recombinant human IL-6 expressed in E. coli undergoes selective N-terminal degradation: evidence that the protein consists of a stable core and a nonessential flexible N-terminal.

A synthetic gene for human interleukin-6 has been expressed in E. coli. The protein has been purified and renatured and has the same activity as natural human IL-6 using the 7TD1 cell proliferation assay. The protein undergoes specific cleavage by a thiol protease, yielding two new N-termini at Arg-9 and His-15. The truncated proteins retain full biological activity. The degradation results in the loss of sharp amide resonances in the 1H-NMR spectrum, and little change to the ultraviolet CD spectrum. Several amino acid type assignments could be made for these sharp amides using a DQF-COSY 2D-NMR experiment. The N-terminal 15 amino acids exist as a flexible, random coil, attached to a central structure.

Fluorescence techniques for fundamental and applied studies of membrane protein receptors: the 5-HT3 serotonin receptor.

A fluorescently labelled ligand for the 5-HT3 serotonin receptor was synthesised and its sub-nanomolar affinity for the purified, detergent solubilised receptor was measured. The change in the ligand's fluorescence upon receptor binding was used to directly measure its dissociation constant for receptor binding, to determine the pharmacology of the receptor, and finally to characterise the binding site of the receptor. A total internal reflection fluorescence (TIRF) assay for the 5-HT3 receptor was developed, which is suitable for high-through-put screening. Therefore, the receptor was immobilised via its C-terminal His-tag onto a nitrilotriacetic acid-modified quartz surface. The affinities of both the fluorescent ligand and several non-fluorescent compounds were rapidly determined by the TIRF assay, and were shown to agree well with both the solution and classical radioligand binding assays. This indicated that the functional integrity of the receptor was preserved at the sensor surface. Due to the extreme sensitivity of the TIRF assay allows to obtain a complete pharmacological affinity profile of a quantity of receptor provided by a small number of highly-expressing cells.

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System.

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT(3) receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix(TM) agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 10(6) cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT(3) receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.

Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system.

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinant Drosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by the Drosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.

Soluble interleukin-5 receptor alpha-chain binding assays: use for screening and analysis of interleukin-5 mutants.

Interleukin-5 (IL-5) is a key cytokine for the production, differentiation, and activation of eosinophils. IL-5 is a member of the four helical bundle family of cytokines, and in common with many members of the cytokine family it binds to a heterodimeric receptor composed of a ligand binding alpha-chain and a signal-transducing beta-chain. We have established two receptor/ligand binding assays based on the extracellular domain of the receptor alpha-chain which we have produced as a fusion protein. One assay is based on scintillation proximity fluoromicrospheres and radiolabeled ligand and the other on detection of biotinylated ligand binding to immobilized receptor using a chemiluminescent substrate in a 96-well microtiter plate format. Both receptor binding assays have been optimized for high throughput screening for receptor antagonists. These assays were also used for analytical purposes and the binding of ligand to the receptor alpha-chain was compared directly to receptor binding assays performed on TF-1 cells which express the receptor alpha beta-heterodimer. These three assays have been used to study site-directed mutants of IL-5 to determine the important residues for interaction of the cytokine with each chain of the receptor (P. Graber et al. (1995) J. Biol. Chem. 270, 15762-15769).

The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution.

Phosphomannose isomerase (PMI) catalyses the reversible isomerization of fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P). Absence of PMI activity in yeasts causes cell lysis and thus the enzyme is a potential target for inhibition and may be a route to antifungal drugs. The 1.7 A crystal structure of PMI from Candida albicans shows that the enzyme has three distinct domains. The active site lies in the central domain, contains a single essential zinc atom, and forms a deep, open cavity of suitable dimensions to contain M6P or F6P The central domain is flanked by a helical domain on one side and a jelly-roll like domain on the other.

Purification, characterisation and crystallisation of selenomethionyl recombinant human interleukin-5 from Escherichia coli.

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.