Secretory production of hGM-CSF with a high specific biological activity by transgenic plant cell suspension culture

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM-CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 μg/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 μg/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation fromE. coli.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Bioreactor operation for transgenic Nicotiana tabacum cell cultures and continuous production of recombinant human granulocyte-macrophage colony-stimulating factor by perfusion culture

The production of hGM-CSF was investigated in both a flask and a 5-l bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g l⁻¹ and 6.5 μg l⁻¹, respectively, those in the bioreactor were 15.6 g l⁻¹ and 7.6 μg l⁻¹. No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day⁻¹). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 μg accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g l⁻¹ of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes.     

IgG stability in fresh and conditioned medium of tobacco (Nicotiana?tabacum) and larch (Larix?decidua) embryogenic suspension cultures

To explore the feasability of larch (Larix decidua Mill.) embryogenic cell culture as alternative plant expression system, protein stability in fresh and conditioned medium was characterized in this study and compared to tobacco BY2 suspension culture. Fresh and conditioned media were spiked with 1 μg human IgG and IgG content was determined by ELISA after 24 h incubation. In fresh media, IgG recovery rate decreased to 12–23%. Adsorption on vessel walls probably is the best explanation for this IgG loss and EDTA in the medium strongly influenced wall adsorption. A high IgG recovery rate occurred in all conditioned cell culture media (7 or 14 days after inoculation). Changes in the low molecular weight-constitution of conditioned medium, rather than co-secreted polymers, are responsible for IgG stability in the cell suspension cultures.     

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.     

Protease activity in protein-free NSO myeloma cell cultures

Summary Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45–50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30–35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5–4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.     

Regulation of extracellular protease formation by Serratia marcescens.

Heavy cell suspensions of Serratia marcescens, when grown in gelatin-containing media, produce extracellular proteases which increase in specific activity in a linear fashion for 3 to 4h. During partial purification, a single peak of proteolytic activity was demonstrated by Sephadex G-100 chromatography. However, electrophoresis using 5% polyacrylamide gels discloses three proteolytically active bands. Evidence in favor of gelatin acting as an inducer of the 'proteolytic system' was provided by two observations. First, proteolytic activity is only present in media containing gelatin. Secondly, the addition of 10(-4) M rifampicin to cells growing in gelatin-containing medium plus an additional carbon source inhibits protease activity totally, but has no effect on growth. When glycerol is added to a growing cell suspension in gelatin-containing medium, growth increases, but protease specific activity decreases. This 'glycerol effect' is not due to an accumulation of active or inactive enzyme in association with the cell, nor to a decrease in the total number of proteases synthesized. Rather, glycerol, as other utilizable carbohydrates, exerts a repression which can be eliminated by 5 mM dibutyryl cyclic AMP.     

Influence of the culture medium composition on the excreted/secreted proteases from <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis>

Streptomyces violaceoruber produces two different classes of mycelium, the substrate and the aerial mycelium. Since proteases have been associated with morphological turnover processes in other Streptomyces species, the presence of excretory/secretory proteolytic activities was investigated here in S. violaceoruber culture supernatants. Various polypeptide bands, with apparent molecular masses ranging from 40 to 180 kDa, were detected in soy trypticase broth (STB) culture media supernatants following 72 h of growth, using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Zymograms showed the presence of five proteolytic enzymes (Spvio1–5), which migrated as bands of 167.7, 130.7, 110.7, 48.3 and 40.9 kDa, respectively. The characterization of these proteases by specific inhibitors showed that Spvio1–4 belong to the serine protease group and Spvio5 corresponds to a cysteine protease. Additionally, Spvio2 and 5 were inhibited by a mixture of EDTA and EGTA, indicating that both require divalent cations. The protease pattern obtained in STB enriched with glucose was identical to that obtained in STB. However, Spvio3 and 4 were absent when nitrogen was added to the culture medium. Cell death was fluorescently detected following 72 h of S. violaceoruber growth in STB and in STB that was enriched with glucose. On the contrary, no cell death was detected in nitrogen-enriched STB media. Additionally, the formation of the aerial mycelium was impaired in solid cultures of STB media enriched with nitrogen. These results demonstrate that the composition of the media influences the morphological turnover of the colony and the pattern of excreted/secreted proteases from S. violaceoruber, and suggest that Spvio3 and 4 are involved in the aerial mycelium formation.     

Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium

Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing the concentration of HC to 10 μg/ml (2.8×10⁻⁵ M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10⁻⁵ M putrescine, 10⁻⁵ M vitamin B₁₂, or 3.7×10⁻⁶ M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v) wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus, factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either on clonal growth or on cumulative multiplication potential of HK. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

Protease excretion during pineapple micropropagation in temporary immersion bioreactors

Summary Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.     

Supernatant proteolytic activities of High-Five insect cells grown in serum-free culture

The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.     

Characterization of a novel cysteine peptidase from tissue culture of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary A simple and efficient medium for callus tissue culture from garlic to obtain maximal proteolytic activity is described. Murashige and Skoog basal medium supplemented with 4.44 μM naphthaleneacetic acid (NAA) and 0.54 μM benzyladenine (BA) resulted in the best biomass production and protease expression. The protease activity belongs to the class of cysteine proteases since they are inhibited by E₆₄ and Leupeptin and also they are activated by 2-mercaptoethanol and cysteine. They showed good thermal stability. Three active protease bands were found in zymograms of Allium sativum. The in vitro system revealed a significantly higher protease level than storage and embryo tissues of in vivo bulbs.     

Enhanced production of recombinant proteins from plant cells by the application of osmotic stress and protein stabilization

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was produced from transgenic Nicotiana tabacum cells. The application of osmotic stress through the addition of 90 g/l mannitol to the plant cell medium enhanced the maximum extracellular GM-CSF concentration from 76 g/l to 130 g/l (1.7-fold increase). The addition of bovine serum albumin (BSA), along with mannitol, further increased the maximum extracellular GM-CSF concentration by as much as 2.5-fold over the control. GM-CSF degradation studies in conditioned medium demonstrated that mannitol and BSA both stabilize the GM-CSF protein. The addition of gelatin together with mannitol to the plant cell medium also enhanced the maximum extracellular GM-CSF concentration and stability over time.     

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 μg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 μg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 μM Fe²⁺ to this medium increased the yield to 226 μg/g DCW.     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

Response surface methodology in media optimization for production of β-carotene from <Emphasis Type="Italic">Daucus carota</Emphasis>

Plants are a valuable source of a vast array of chemical compounds including fragrances, flavours, food additives, colours, natural sweeteners, industrial feedstocks, anti-microbials and pharmaceuticals. The present study reports on application of Response Surface Methodology (RSM) in media optimization for suspension culture for the production of β-carotene. Growth kinetics of carrot cells in suspension culture has been carried out to understand the relationship between growth and β-carotene formation. The maximum production of β-carotene obtained using the optimized medium was 13.614 μg/g dry weight cell mass. The μ (specific growth rate) and t _d (doubling time) were found to be higher for 20 g DW/l inoculum size.     

Increased production of laccase by <Emphasis Type="Italic">Cerrena unicolor</Emphasis> in submerged liquid cultures

The wood-degrading basidiomycete Cerrena unicolor C-139 has been suggested as a potential producer of the industrially important enzyme laccase. Basic culture parameters influencing the enzyme synthesis in shaken-flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably enhanced by the addition of Cu²⁺ in the micromolar range to a carbon-sufficient and nitrogen-sufficient culture medium (C/N = 16.69). When an optimised medium containing glucose (10 g/L) and l-asparagine (1.5 g/L) was used, and enzyme synthesis was stimulated by addition of 10 μM Cu²⁺ to the culture medium on days 3, 6 and 9, maximal laccase productivity obtained after 17 days’ cultivation in shaken flask cultures was above 100,000 nkat/L. In fermenter fungal cultures, the influence of stabilisation of medium pH on laccase activity was additionally studied. The use of a bioreactor with an automatic pH control set at pH 6.5 after 48-h incubation resulted in the enzyme activity of 65,000 nkat/L after 8 days’ cultivation.     

Protease expression in the supernatant of Chinese Hamster Ovary cells grown in serum-free culture

The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B.     

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.