Improvement of plastic-based disposable bioreactors for plant science needs

The present article describes two new applications of plastic-based cell culture systems in the plant biotechnology domain. Different types of bioreactors are used at Nestlé R&D Center-Tours for large scale culture of plants cells to produce metabolites or recombinant proteins and for mass propagation of selected plant varieties by somatic embryogenesis. Particularly, recent studies are directed to cut down the production costs of these two processes by developing disposable cell culture systems. For large scale culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 l working volumes, validated with several plant species (“Wave and Undertow” and “Slug Bubble” bioreactors). Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has been recently set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 2.5–3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-l glass bioreactors. An improved process has been developed using a 10-l disposable bioreactor consisting in a bag containing a rigid plastic box (“Box-in-Bag” bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design.     

Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli

In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225′-232′), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.This work was supported by the National High Technology “863” Programs of China (no. 2002 AA217031-2), a Grant-in-Aid from China National Natural Science Fund Committee (grant no. 30270298) and Jiangsu Natural Science Fund Committee (grant no. BK 95092309 and BG2001011).     

Process development for production of recombinant human insulin-like growth factor-I in Escherichia coli

Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production. Under this condition, 2.8 g L⁻¹ of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography, refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99. Received 13 January 1999/ Accepted in revised form 02 October 1999     

A modified hepatitis B surface antigen carrying both preS1 and preS2 epitopes

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Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves. An erratum to this article can be found at     

Production of Bioactive γ-Glutamyl Transpeptidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Using SUMO Fusion Partner and Application of the Recombinant Enzyme to <Emphasis Type="SmallCaps">l</Emphasis>-Theanine Synthesis

The amino acid l-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of l-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni–NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of l-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of l-theanine.     

Effects of calcium ion on adenovirus production with high densities of HEK293 cells

One of the major restrictions on the production of adenoviral vectors (AdV) is due to the decrease in virus concentration concomitant with an increase in cell concentration at infection (CCI) which is known as “cell density effect”, this could be because of the limited access to the nutrients or significant accumulation of toxic by-products. However, current strategies, such as developing robust serum-free medium and performing nutrient feeding, will partially address this issue. Therefore, the objective of this study was to further optimize serum-free culture medium by exploring the influence of calcium ion on virus production. Our studies showed that an optimal concentration of calcium ion significantly enhances AdV production, especially at a high CCI. During the virus infection process, a high concentration of calcium ion (≥ 1 mM) caused a reduction in virus infection efficiency, possibly as a result of cell aggregation. However, by optimizing the timing of the addition of calcium ion (i.e., 12 hours post-infection [hpi]), high virus infection efficiency could be maintained. The addition of 0.1 to 2 mM calcium ion at 12 hpi increased virus proliferation dose-dependently. Therefore, the optimal selection of both the concentration and the supplementation time of calcium ion during the process of AdV production could minimize the “cell density effect,” and a 2.6- and 3.2-fold increase in virus concentration could be achieved at CCI3 and CCI4, respectively.     

Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.     

Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system.     

Tapping culture-an improved method for cell suspension culture

Summary A new culture vessel was designed for cell suspension culture. A silicone-convered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of “stirring” in cultures the bar moves vertically with a “tapping” motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures than in stirring cultures. Serial cultivation of cells in tapping cultures was also successful. This work was supported in part by the grants for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

Md-ACS1 and Md-ACO1 genotyping of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis> Borkh.) breeding parents and suitability for marker-assisted selection

Fruit ethylene production genotypes for Md-ACS1 and Md-ACO1 were determined for 60 apple cultivars and 35 advanced breeding selections. Two alleles for each gene are commonly found in cultivated apple. Earlier studies showed that genotypes homozygous for the ACS1-2 allele produce less ethylene and have firmer fruit than ACS1-1/2 and ACS1-1/1 genotypes. ACO1 plays a minor role compared to ACS1, with homozygous ACO1-1 having lower ethylene production. In this study, ACS1-2 and ACO1-1 homozygotes had firmer fruit at harvest and after 60 days of 0–1°C cold storage compared to other genotypes. These genotypes, ACS1-2/2 and ACO1-1/1, were observed for the following 8 of 95 cultivars/selections: “Delblush”, “Fuji”, “Pacific Beauty”, “Sabina” and four breeding selections. Cultivars/selections that were homozygous ACS1-2 but not ACO1-1 were: “Ambrosia”, “Aurora Golden Gala”, “CrimsonCrisp”, “Gala”, “GoldRush”, “Huaguan”, “Pacific Rose, “Pacific Queen”, “Pinova”, “Sansa”, “Sonja”, “Sundance”, “Zestar”, and 17 breeding selections. Cultivars with the heterozygous ACS1-1/2 genotype were “Arlet”, “Braeburn”, “Cameo”, “Delicious”, “Delorgue”, “Empire”, “Enterprise”, “Ginger Gold”, “Golden Delicious”, “Granny Smith”, “Honeycrisp”, “Orin”, “Pink Lady”, “Silken”, “Suncrisp”, “Sundowner”, “Sunrise” and 11 breeding selections. No cultivars were detected homozygous for both ACS1-1 and ACO1-1, or for both ACS1-2 and ACO1-2. This study is the first large-scale allelic genotyping of both ethylene synthesis genes for a comprehensive set of apple breeding parents used in an ongoing breeding project. The data reported here are important for informative selection of parent combinations and marker-assisted selection of progeny for breeding low ethylene-producing apple cultivars for better storability and improved consumer acceptance.     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation,membrane binding,and electrostatics,and implications for HIV-host cell fusion

The HIV gp41 protein catalyzes fusion between HIV and target cell membranes. The fusion states of the gp41 ectodomain include early coiled-coil (CC) structure and final six-helix bundle (SHB) structure. The ectodomain has an additional N-terminal apolar fusion peptide (FP) sequence which binds to target cell membranes and plays a critical role in fusion. One approach to understanding gp41 function is study of vesicle fusion induced by constructs that encompass various regions of gp41. There are apparent conflicting literature reports of either rapid or no fusion of negatively charged vesicles by SHB constructs. These reports motivated the present study, which particularly focused on effects of pH because the earlier high and no fusion results were at pH 3.0 and 7.2, respectively. Constructs include “Hairpin,” which has SHB structure but lacks the FP, “FP-Hairpin” with FP + SHB, and “N70,” which contains the FP and part of the CC but does not have SHB structure. Aqueous solubility, membrane binding, and vesicle fusion function were measured at a series of pHs and much of the pH dependences of these properties were explained by protein charge. At pH 3.5, all constructs were positively charged, bound negatively charged vesicles, and induced rapid fusion. At pH 7.0, N70 remained positively charged and induced rapid fusion, whereas Hairpin and FP-Hairpin were negatively charged and induced no fusion. Because viral entry occurs near pH 7 rather than pH 3, our results are consistent with fusogenic function of early CC gp41 and with fusion arrest by final SHB gp41.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l⁻¹ and 7 g fusion glucagon l⁻¹. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content. Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997     

Design of bioreactors suitable for plant cell and tissue cultures

Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In contrast to traditionally grown “whole wild plants” or “whole transgenic plants”, their production in bioreactors guarantees defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing acceptance over the last 10 years. Plants for human health in the post-genome era, PSE congress 26.8.2007–29.8.2007, Helsinki.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

Hollow-fibre bioreactors compared to batch and chemostat culture for the production of a recombinant toxoid by a marine Vibrio

Bioreactor selection is important for maximising the productivity of recombinant organisms. In this paper a comparison is made between growth and recombinant protein synthesis in three types of bioreactor containing a marine Vibrio capable of heterologous expression and secretion of the non-toxic B-subunit pentamer of Escherichia coli heat-labile enterotoxin, EtxB. The heterologous gene was located on the plasmid pMMB68. Resistance to carbenicillin was used to select for plasmid-containing cells. In batch and continuous culture, volumetric productivities were highest when cells were grown in the presence of carbenicillin. Without antibiotic selection, the highest volumetric productivity (9.4 mg EtxB⁻¹ h⁻¹) was observed in hollow-fibre bioreactors, and the production phase could be maintained for over 50 h. The highest specific productivity under these conditions was found in batch culture, but the maximal production phase was only of 5 h duration. In hollow-fibre reactors the type of fibre used significantly affected productivity, both with regards to the maintenance of reactor integrity and by allowing passage of the recombinant toxoid through the selectively permeable membrane. Where contamination of the product with carbenicillin is to be avoided, these bioreactors are superior to batch or continuous culture. Received: 29 January 1997 / Received revision: 9 April 1997 / Accepted: 13 April 1997     

Perstraction of intracellular pigments by submerged cultivation of <Emphasis Type="Italic">Monascus</Emphasis> in nonionic surfactant micelle aqueous solution

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.     

Overproduction of soluble, extracellular cytotoxin α-sarcin inEscherichia coli

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin α-sarcin to be used for immunoconjugate production. α-Sarcin cDNA was isolated fromAspergillus giganteus strain MDH 18894 and its expression inEscherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant α-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernant. A variant form of α-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.