Hideki Yamaji

Use of nonionic surfactants for effective supply of phosphatidic acid in serum-free culture of chinese hamster ovary cells

We have previously shown [Sakai et al., J. Biosci. Bioeng., 88, 306-309 (1999)] that exogenously supplied phosphatidic acid (PA) and lysophosphatidic acid (LPA) promoted the growth of Chinese hamster ovary (CHO) cells in serum-free culture. However, the direct addition of high concentrations of these phospholipids alone to the culture medium resulted in the formation of precipitates. We therefore examined the use of two nonionic surfactants, Tween 80 and Pluronic F-68, as a means of supplying PA more effectively to CHO cells in a serum-free culture. A clear dispersion of PA from egg yolk lecithin that could be successfully sterile-filtered was obtained by using Tween 80 or Pluronic F-68. When PA prepared with either of the surfactants was added to serum-free media, precipitation was noticeably reduced. Furthermore, the growth-promoting activity of PA was considerably enhanced by the presence of the surfactants. Since Tween 80 and Pluronic F-68 themselves possessed no growth-stimulating property, it was suggested that the enhanced growth-promoting activity results from the improved availability of PA to the cells. The use of Tween 80 with PA analogues having saturated acyl chains also accelerated cell growth, whereas these PAs showed little growth-promoting activity, due to their poor water-solubility, when added alone.

Immobilisation of anchorage-independent animal cells using reticulated polyvinyl formal resin biomass support particles

SummaryImmobilisation of anchorage-independent animal cells using Biomas Support Particles (BSPs) was investigated. Mouse myeloma MPC-11 cells were physically entrapped in three-dimensional reticulated polyvinyl formal (PVF) resin PSPs (3×3×3 mm) with matrices of relatively small pores (30–100 μm) by filtering medium containing cells or incubating in a shake flask for inoculation. Physically entrapped cells became immobilised in the BSPs by forming aggregates within the matrices of the reticulated PVF resin, and cell density in the BSPs reached at least 107 cells/cm3 BSP. Immobilised cells in the BSPs were successfully cultivated in static and/or shake-flask cultures with regular replacement of medium for a long period.

High efficiency production of astaxanthin in an airlift photobioreactor.

In photobioreactors, photosynthetic microorganisms are exposed to certain light/dark cycles caused by light intensity distribution and mixing inside the photobioreactor. In this study, Haematococcus pluvialis was cultivated in an airlift and a bubble column photobioreactor, and the cell growth and astaxanthin production were compared to clarify the effects of liquid circulation.

Angiotensin-I Converting Enzyme (ACE) Inhibitory Mechanism of Tripeptides Containing Aromatic Residues

Angiotensin-I converting enzyme (ACE) plays important roles in the regulation of blood pressure, and ACE inhibitory peptides in food materials have attracted attention for their antihypertensive function. In this study, the function of amino acids in ACE inhibitory tripeptides was clarified.

Optimal production of recombinant protein by the baculovirus-insect cell system in shake-flask culture with medium replacement

Sf9 insect cells infected with a recombinant baculovirus expressing beta-galactosidase and suspended in fresh medium (TNM-FH supplemented with 10% fetal bovine serum) at the time of infection were cultured in shake flasks at various combinations of initial cell density and multiplicity of infection (MOI). The effects of cell density and MOI on beta-galactosidase production were quantitatively analyzed by plotting the beta-galactosidase yield against the time integral of the viable cell density from the time of infection to the time when the beta-galactosidase production reached a plateau. The beta-galactosidase yield had a maximum value at a viable cell density time integral of approximately 8 x 10(6) cells.d/cm3 for each MOI used in a range from 0.01 to 10 plaque-forming units per cell (pfu/cell). Since glucose and fructose were exhausted when the culture reached 8 x 10(6) cells.d/cm3, it was concluded that protein production in a high-cell-density culture was limited by nutrient depletion in the culture medium, and hence the nutritional capacity of the medium was able to be determined as the viable cell density time integral at which the maximum product yield was attained. In cultures infected at a low MOI (< or =1 pfu/cell), the specific productivity, and thereby the yield, of beta-galactosidase declined with decreasing MOI due to the reduction in the proportion of initially infected cells. These results indicate that production of a recombinant protein in a culture with medium replacement at the time of infection can be optimized if the cells are infected at a high MOI (> or = 1 pfu/cell) and at a cell density such that the viable cell density time integral reaches the nutritional capacity just as the protein production is completed.

Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli

The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective.

A simple method for measuring the complement activities of both classical and alternative pathways by using rabbit γ-globulin-coupled liposomes

Abstract Rabbit γ-globulin (RGG) was covalently coupled to liposomes in which carboxyfluorescein was encapsulated as a marker. Lysis of the liposomes was observed under incubation with guinea pig complement. However, no lysis was observed with heat-inactivated complement. These results suggest that lysis of the liposomes is caused by the activation of complement with coupled RGG in particular, through the alternative parhway of complement (APC). By addition of anti-rabbit IgG antibodies to the RGG-coupled liposomes, lysis of the liposomes was remarkably enhanced. This may be due to the additional activation of the classical pathway of complement (CPC) by antigen-antibody complexes formed on the surface of liposomes. Thus, the activities of both the pathways can be measured easily in the same system by using RGG-coupled liposomes.

Effects of temperature on the astaxanthin productivity and light harvesting characteristics of the green alga Haematococcus pluvialis.

The green alga Haematococcus pluvialis, which accumulates astaxanthin at an optimal temperature of 20°C, was cultivated under temperatures of 20°C, 23.5°C, 27°C, and 30.5°C, in order to assess the effects on algal metabolism during the growth phase. The culture growth rate declined with above-optimal increases in temperature, and the final maximum cell concentration at 30.5°C reached only 35% of that attained at 20°C. On the contrary, the biomass productivity was increased under all the high-temperature conditions, probably reflecting the metabolism switch from cell duplication to energy accumulation that is typically observed in algal cultures subjected to environmental stress. Moreover, an increase in the light-harvesting capability of the alga was observed by means of the total pigment balance and the photosynthesis-intensity (PI) curve measured under the different cultivation conditions. Cultures kept at higher temperatures were able to better harvest and utilize the impinging light due to photo-acclimation. Finally, the differences in the astaxanthin metabolism were elucidated by subjecting the cultures to nitrogen starvation at 20°C and 27°C. In the culture at 27°C, a 1.4-fold increase in the astaxanthin productivity was observed when compared to that at 20°C, and the latter required almost two-fold more energy for the astaxanthin production compared with the 27°C culture.

Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones

The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expression of functional scFvs is increased by co-expression of molecular chaperones. In the present study, we examined the effects of the combination of Trx fusion and molecular chaperone co-expression on the production of a functional scFv. A Trx-fused scFv was obtained in the oxidizing cytoplasm, and co-expression of GroELS and trigger factor had the greatest effect, resulting in a 2.8-fold increase in specific productivity. By contrast, the molecular chaperone DnaKJE had no effect. Moreover, co-expression of DnaKJE with GroELS negated the effects of GroELS. Trx-scFv was purified using a bovine ribonuclease A-coupled Sepharose column, and 2.7 mg/L of purified protein was obtained. Soluble Trx-scFv, expressed and purified as described above, exhibited pH-dependent binding similar to that of the parental full-length antibody. In addition, approximately 80% of the initial binding activity was retained after incubation at 37 degrees C for 2 weeks, indicating that the Trx-scFv fusion protein is quite stable. This strategy might be useful for the preparation of other recombinant scFvs.

Production of Japanese encephalitis virus-like particles using the baculovirus-insect cell system.

The production of a secreted form of Japanese encephalitis (JE) virus-like particles (VLPs) using the baculovirus-insect cell system was investigated. A recombinant baculovirus that contained the JE virus (JEV) prM signal sequence and the genes encoding the precursor (prM) of the viral membrane protein (M) and the envelope glycoprotein (E) was constructed. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the culture supernatant showed that Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus had secreted the E protein. Sucrose density-gradient sedimentation analysis of the culture supernatant suggested that secreted E antigen molecules were in a particulate form. Baculovirus-infected Sf9 cells produced more than a 10-fold higher yield of E antigen than that produced by previously reported recombinant CHO cells. Following infection with a recombinant baculovirus encoding a form of prM with a pr/M cleavage site mutation designed to suppress cell-fusion activity of E, Sf9 cells showed an E antigen yield comparable to a yield obtained with the baculovirus encoding the authentic form of prM. Baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted less of the E antigen than Sf9 cells. Moreover, the Drosophila BiP signal sequence gave an E antigen yield comparable to the prM signal sequence, while the honeybee melittin signal sequence and the baculovirus gp64 signal sequence resulted in lower yields of the E antigen. These results provide information important to the development of VLP production processes using the baculovirus-insect cell system.