Plant Cell Suspension Cultures Research Articles

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.

L-Alanine induces programmed cell death in V. labrusca cell suspension cultures

Plant cell suspension cultures are reliable year-round sources of enzymes involved in secondary metabolite biosynthesis. Numerous studies have used V. vinifera cell suspension cultures as an experimental system for studying these processes, while those from Concord grape ( V. labrusca L.) have been poorly exploited. V. vinifera cell suspension cultures have been shown to grow well with casein hydrolysate (CH), which is a good source of reduced nitrogen in many tissue culture systems. The present report shows that V. labrusca cell suspension cultures, in contrast to those of V. vinifera, could only be established in the absence of CH as a source of reduced nitrogen. CH contains 20 amino acids, among which, the single amino acid l-alanine activates a programmed cell death response in V. labrusca cells, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay and propidium iodide nuclei staining. Programmed cell death was accompanied by the coordinated activation of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and stilbene synthase (STS) gene expression, as well as by the accumulation of stilbenes and other phenolic compounds.

Elicitation of rosmarinic acid by Lavandula vera MM cell suspension culture with abiotic elicitors

The growth of Lavandula vera MM plant cell suspension culture and rosmarinic acid biosynthesis under elicitation with benzothiadiazole and methyl jasmonate were investigated. Upon elicitation with 50 µM methyl jasmonate, the production of rosmarinic acid was enhanced 2.4-fold (3348 mg/l) compared to the non-elicited cells. The influence of benzothiadiazole on rosmarinic acid biosynthesis was weaker and 12 h after its addition the achieved yields were 20–30% higher compared to the control variant at this time. The influence of both elicitors on rosmarinic acid secretion into the culture medium was also discussed.

Phenylpropanoid metabolite supports cell aggregate formation in strawberry cell suspension culture

Plant cells in suspension culture tend to aggregate and form large clumps. In suspension culture, large cell aggregates are frequently subjected to hydrodynamic shear stress; however, a certain degree of cell aggregation is often required for cell growth and metabolite production. Thus, controlling cell-aggregate size is desired to establish high productivity of useful products using plant cell suspension culture. In this study, we focused on the relationship between cell-aggregate formation and secondary metabolism. We found that anthocyanin concentration showed a good correlation with cell-aggregate size in the cultured strawberry cell line FAR (Fragaria ananassa R), which produces anthocyanin and other phenylpropanoid metabolites constitutively without illumination. This result suggests that there is a relationship between cell-aggregate formation and the accumulation of phenylpropanoid metabolites. To investigate the direct effect of phenylpropanoid metabolism on cell-aggregate formation, the time course of cell-aggregate size was monitored when phenylpropanoid metabolism was suppressed by a metabolic inhibitor, L-alpha-aminooxy-beta-phenylpropionic acid (AOPP), a specific inhibitor of phenylalanine ammonia lyase which is the starting and key enzyme of the phenylpropanoid pathway. In the absence of AOPP, the average diameter of cell aggregates increased on day 8 of culture. This increase in cell-aggregate size was completely suppressed by the addition of 0.1 mM AOPP, without any reduction in cell growth rate or soluble protein content. These results indicate that cell-aggregate formation is directly supported by a secondary metabolite produced from the phenylpropanoid pathway, suggesting that cell-aggregate size can be controlled by AOPP without inhibition of primary metabolism.

Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering

Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol ®) for the treatment of many cancers. To develop an optimal bioprocess for paclitaxel supply, taxane biosynthetic pathway regulation must be better understood. Here we examine the expression profile of paclitaxel biosynthetic pathway genes by RNA gel blot analysis and RT-PCR in the Taxus cuspidata cell line P991 and compare with taxane metabolite levels. Upon methyl jasmonate (MJ) elicitation (100 μM), paclitaxel accumulates to 3.3 mg/L and cephalomannine to 2.2 mg/L 7 days after elicitation but neither are observed before this time. 10-deacetylbaccatin III accumulates to 3.3 mg/L and baccatin III to 1.2 mg/L by day 7 after elicitation. The early pathway enzyme genes GGPPS, TASY, and T5αH are up-regulated by MJ elicitation within 6 h and continue through 24 h before their abundances decrease. This study reveals the preference for one side of the biosynthetic pathway branch in early taxane synthesis, where transcripts coding for T αH are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch ( TDAT and T10βH) are not highly expressed following elicitation. Transcripts encoding the enzymes DBBT and DBAT are up-regulated upon MJ elicitation. Their products, 10-deacetylbaccatin III and baccatin III, respectively, accumulate within 6 h of the initial increase in transcript abundance. Importantly, the steady-state levels of the two terminal enzyme transcripts ( BAPT and DBTNBT) are much lower than transcripts of early pathway steps. These are potential steps in the pathway for targeted metabolic engineering to increase accumulation of paclitaxel in suspension cell culture.

Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 μg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.

Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells

Although suspension-cultured plant cells have many potential merits as sources of useful proteins, the lack of an efficient expression system has prevented using this approach. In this study, we established an inducible tomato mosaic virus (ToMV) infection system in tobacco BY-2 suspension-cultured cells to inducibly and efficiently produce a foreign protein. In this system, a modified ToMV encoding a foreign protein as replacement of the coat protein is expressed from stably transformed cDNA under the control of an estrogen-inducible promoter in transgenic BY-2 cells. Estrogen added to the culture activates an estrogen-inducible transactivator expressed constitutively from the transgene and induces transcription and replication of viral RNA. In our experiments, accumulation of viral RNA and expression of green fluorescent protein (GFP) encoded in the virus were observed within 24 h after induction. The amount of GFP reached approximately 10% of total soluble protein 4 d after induction. In contrast, neither viral RNA nor GFP were detected in uninduced cells. The inducible virus infection system established here should be utilized not only for the expression of foreign proteins, but also for investigations into the viral replication process in cultured plant cells.

Increase in peroxidase activity in tea callus in response to darkness, 2,4-dichlorophenoxyacetic acid and carbohydrates

The peroxidase (POD) reaction for the industrial synthesis of etoposide proceeds effectively when suspension-cultured plant cells are used as catalysts. Tea (Camellia sinensis) suspension cultures express higher POD activity and produce a higher yield of reaction products than those of other plant cells. We intend to investigate the conditions necessary to efficiently increase the POD activity in a suspension-cultured tea callus. Here we examined the influence of photoenvironmental conditions, phytohormones, and carbohydrates on POD activity in the callus grown on solidified medium. POD activity in the callus was increased 1) in the dark; 2) on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 10 mg l−1; or 3) on medium containing 0.3 M sucrose and 0.1 M mannitol. The highest POD activity, achieved under the third set of conditions, was about 3.6-fold higher than that achieved on control medium (with 0.09 M sucrose).

Magnetic field exposure stiffens regenerating plant protoplast cell walls

Single suspension-cultured plant cells (Catharanthus roseus) and their protoplasts were anchored to a glass plate and exposed to a magnetic field of 302 +/- 8 mT for several hours. Compression forces required to produce constant cell deformation were measured parallel to the magnetic field by means of a cantilever-type force sensor. Exposure of intact cells to the magnetic field did not result in any changes within experimental error, while exposure of regenerating protoplasts significantly increased the measured forces and stiffened regenerating protoplasts. The diameters of intact cells or regenerating protoplasts were not changed after exposure to the magnetic field. Measured forces for regenerating protoplasts with and without exposure to the magnetic field increased linearly with incubation time, with these forces being divided into components based on the elasticity of synthesized cell walls and cytoplasm. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye, and no changes were noted after exposure to the magnetic field. Analysis suggested that exposure to the magnetic field roughly tripled the Young's modulus of the newly synthesized cell wall without any lag.

Heat-induced oxidative activity protects suspension-cultured plant cells from low temperature damage.

A short heat pre-treatment (1 h at 38°C) was found to protect both suspension-cultured apple fruit cells and tobacco cells from cold-induced cell death. Tobacco cells were more sensitive to low temperatures than apple cells, with significant cell death after 48 h at 0 or -2°C. Real-time measurements of H2O2 levels during the heat pre-treatment revealed a substantial burst of this reactive oxygen species in both cell types. Real-time and longer-term measurements also showed a large burst of H2O2 production from tobacco cells, but not apple cells, when exposed to low temperatures. Lower temperatures reduced levels of peroxidase activity (both total and intracellular), with the heat pre-treatment preventing some of the cold-induced reduction of this activity in both apple and tobacco cells. The greater sensitivity to low temperature of the tobacco cells may be related to higher H2O2 production, with the heat treatment maintaining higher peroxidase activity. The lesser sensitivity of the apple cells may be due to the lack of a H2O2 burst and maintenance of peroxidase activity by the heat treatment. These results support a role for oxidative metabolism in the beneficial effects of heat in inducing low temperature tolerance.

Modeling of azadirachtin production by Azadirachta indica and its use for feed forward optimization studies

Azadirachtin, obtained from Azadirachta indica (neem) has emerged as wonderful natural biopesticide for pest control due to its efficacy, biodegradability and minimum side effects. To overcome constraints associated with its limited, variable and unreliable supply from natural trees, plant cell suspension culture of A. indica was developed (obtained from callus of seeds). The kinetics of cell growth/azadirachtin formation and substrate consumption of A. indica suspension culture was established in bioreactor with low shear setric impeller under optimized culture conditions (statistically optimized independently). The culture featured a growth of 15.5 and 0.05 g/l azadirachtin in 10 days of cultivation after which azadirachtin concentration declined. To prevent any inhibition caused by the excess feeding of substrate during fed-batch cultivation, inhibition of the carbon, nitrate and phosphate were separately established. Based on batch kinetics/inhibitory data a mathematical model was proposed and model parameters were evaluated. The model was extrapolated to fed-batch cultivation and nutrient feeding strategy (with respect to carbon, nitrate and phosphate) were computer simulated for a suitable fed-batch cultivation of A. indica cells to enhance growth and product formation. Fed-batch cultivation demonstrated a growth of 20.06 g/l biomass and 0.082 g/l of azadirachtin in 14 days of cultivation. A 30% increase in cell growth and 83% increase in azadirachtin production were obtained when a computer simulated feeding strategy was experimentally implemented in 3 l bioreactor.

The alc-GR System. A Modified alc Gene Switch Designed for Use in Plant Tissue Culture

The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.

Observer‐based online compensation of inner filter effect in monitoring fluorescence of GFP‐expressing plant cell cultures

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.

Role of biotechnology in medicinal plants

Medicinal plants are the most important source of life saving drugs for the majority of the world's population. The biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants. In-vitro regeneration holds tremendous potential for the production of high-quality plant-based medicine. Cryopreservation is long-term conservation method in liquid nitrogen and provides an opportunity for conservation of endangered medicinal plants. In-vitro production of secondary metabolites in plant cell suspension cultures has been reported from various medicinal plants. Bioreactors are the key step towards commercial production of secondary metabolites by plant biotechnology. Genetic transformation may be a powerful tool for enhancing the productivity of novel secondary metabolites; especially by Agrobacterium rhizogenes induced hairy roots. This article discusses the applications of biotechnology for regeneration and genetic transformation for enhancement of secondary metabolite production in-vitro from medicinal plants. Key words: Bioreactors; genetic transformation; regeneration; secondary metabolites Abbreviations: BA: 6-Benzylaminopurine; TDZ: 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea; NAA: a-Naphthaleneacetic acid; IAA: Indole-3 acetic acid; 2iP: 6-(g-Dimethylallylamino) purine; 2,4-D: 2,4-Dichlorophenoxyacetic acid; GA3: Gibberellic acid Trop J Pharm Res , December 2003; 2(2): 243-253

Effect of Subculture and Elicitation on Instability of Taxol Production in Taxus sp. Suspension Cultures

The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.

Introductory Remarks on Biosynthesis and Diversity in Actions

In the early sixties jasmonic acid methyl ester (JAME) was identified as the oil-constituent of jasmin, and the free acid (JA) was first found in the culture filtrate of Botryodiplodia theobromae. However, only in 1980/81 did jasmonates become interesting after Sembdner s group in Halle/Saale (Germany) observed the growth-inhibition of jasmonates and Ueda s group in Osaka (Japan) described the senescence-promoting effect of jasmonates. In the middle of the eighties the biosynthetic pathway for JA was described by Vick and Zimmermann in Fargo (USA). First, jasmonate-induced alteration of gene expression was observed with barley leaves, where several proteins accumulated abundantly upon jasmonate treatment as described by Parthier s group in Halle/Saale in 1986. Since 1990, when Ryan s lab in Pullman (USA) recognized that airborne JAME is a signal for wound-induced proteinase inhibitor gene expression in tomato, numerous sets of genes were identified as being up-regulated or down-regulated by JA. The next breakthrough was the observation in Zenk s lab in Munich (Germany) that external stimuli such as elicitors applied to plant cell suspension cultures led to an endogenous increase of jasmonates followed by alkaloid synthesis. Subsequently, JA was found to be a signal in numerous abiotic and biotic stress responses and in various developmental processes. Some of them are summarized in Figure 1. In the last decade, an enormous interest in JA has appeared, reflected in an exponentially growing number of publications. Some highlights of that period are (i) Identification of mutants of Arabidopsis affected in jasmonate biosynthesis or signalling and being male sterile. (ii) Identification of COI1 which is affected in the JA insensitive mutant coi1 as one of the first Fbox proteins of the ubiquitin/proteasome pathway found in plants (iii) The synergistic and antagonistic signalling of jasmonate, ethylene and salicylate (iv) Identification of the first transcription factors (ORCAs) that regulate JA-responsive gene expression. (v) The role of jasmonate in plant-plant and plant-insect interactions. Among other aspects, all these highlights will be discussed in the present special issue of JPGR. The most recent research on jasmonates is reviewed by experts from the corresponding fields. The 7 papers cover chemistry, physiology, molecular biology and genetics as well as applied research on jasmonates. Several aspects of JA research were reviewed in the last years. The goal of this special issue on jasmonates is to combine the numerous aspects in a parallel and new presentation. Received: 4 October 2004; accepted: 5 October 2004; Online publication: 27 January 2005 *Corresponding author; e-mail: cwastern@ipb-halle.de g ,

Stimulating the production of homoisoflavonoids in cell suspension cultures of Caesalpinia pulcherrima using cork tissue

It has previously been demonstrated that cork tissue increases the efficiency of the production of lipophilic secondary metabolites in diverse plant cell suspension cultures. In the present study, three new homoisoflavonoids – named dihydrobonducellin, 2′-methoxydihydrobonducellin, and 2′-methoxybonducellin – and bonducellin and isobonducellin were isolated from Caesalpinia pulcherrima cultured cells coincubated with cork tissue. Cork tissue increased the production of 2′-methoxybonducellin by about 7-fold relative to control cells, and more than 80% of the product was recoverable from the cork tissue. When cork tissue and methyl jasmonate or yeast extract were added simultaneously to the medium, the amount of 2′-methoxybonducellin produced increased further. The production of the other four homoisoflavonoids was enhanced by variable amounts. Our results indicate that the addition of cork tissue would be an effective technique for investigating formation of secondary metabolites that usually accumulate only in trace amounts.

Growth behavior in plant cell cultures based on emissions detected by a multisensor array.

The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.

Biosynthesis and characterization of 14C-enriched flavonoid fractions from plant cell suspension cultures.

A range of radiolabeled anthocyanins, proanthocyanidins, and other flavonoids were accumulated by cell suspension cultures of two plant species, ohelo (Vaccinium pahalae) and grape (a Vitis hybrid, Bailey Alicant A), after providing uniformly labeled [(14)C]sucrose to the medium. Approximately 15% of administered label was recovered in a series of flavonoid-rich fractions varying in composition. Anthocyanins, and monomers to oligomers of proanthocyanidins, were labeled effectively and characterized from both species. Most of the proanthocyanidin oligomers were based on the flavan-3-ols (+)-catechin and (-)-epicatechin. Cyanidin and peonidin glycosides were the dominant forms of anthocyanins in both species. Whereas the predominant form of flavonoids identified from ohelo cell cultures was proanthocyanidins, grape cell cultures produced mostly anthocyanins. The labeled phytochemicals were produced for use in subsequent in vivo animal feeding studies to gauge their bioavailability and accumulation in target organs.

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andKhGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andKhGM-CSF of 7.64 after 2 h of incubation at room temperature.