Ken-Ichi Kikuchi

Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors.

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Abstract. Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.

Sorption of mono-carboxylic acids by an anion-exchange membrane

Sorption characteristics of eight mono-carboxylic acids by an anion-exchange membrane were studied. The experiments were carried out as batch determinations of the equilibrated concentrations of chloride and carboxylic acid in the membrane and the solution. The sorption of a carboxylic acid by the ion-exchange membrane rapidly increased with increase in the solution concentration of the carboxylic acid, and was considerably different at different pH. However, these pH-dependent sorption equilibria were successfully represented as one curve in terms of the concentration of the dissociated carboxylic acid ion in the solution. This means that the sorption behavior of carboxylic acids can be explained by the ion exchange between a dissociated carboxylic acid ion and a counter ion on the fixed charge in the membrane. On the basis of the experimental results, the selectivity coefficients of mono-carboxylic acids were determined by a model which considered with dissociation equilibrium of carboxylic acids, material balance in the solution, and electrical neutrality in the membrane. The model successfully explained the observed sorption characteristics for carboxylic acids.

Significant increase in recombinant protein production of a virus-infected Sf-9 insect cell culture of low MOI under low dissolved oxygen conditions

Spodoptera frugiperda Sf-9 insect cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (beta-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the cell growth following viral infection was greatest and beta-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4-8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 cells. These results indicated that for a baculovirus-infected Sf-9 insect cell culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intracellularly only at 5% DO, coinciding with beta-galactosidase production.

High-Level Expression of Recombinant Active Human Renin in Sf-9 Cells: Rapid Purification and Characterization

Recombinant human (rh) renin was expressed in Sf-9 insect cells. Baculovirus-infected Sf-9 cells produced active rh-renin in the late stage of cultivation. The rh-renin was purified after 5 d of culture by two steps of column chromatography. Approximately 0.61 mg of pure rh-renin was obtained from 200 ml of culture medium with a yield of 35.3%.

Sorption of di- and tricarboxylic acids by an anion-exchange membrane

Abstract Sorption equilibria of the dicarboxylic acids, oxalic, succinic, adipic, and malic acids, and the tricarboxylic acid, citric acid, by the strongly basic anion-exchange membrane (SELEMION AMV) were studied to obtain the fundamental data for the development of a model of electrodialysis process to separate those acids. These acids were increasingly sorbed with increasing pH and solution concentration, while the sorption of citric acid reached a maximum at pH about 4, and gradually decreases. Non-dimensional selectivities of ionic species of these acids by the membrane were determined by an ion-exchange model that considered the dissociation of carboxylic acid in the solution, and electro-neutrality in the solution and in the membrane. The selectivities of mono-, di-, and tricarboxylic acids were correlated with their molecular weight as well as those of eight mono carboxylic acids. These results are useful for the general estimation of selectivity of carboxylic acids.

Perfluorocarbon-mediated aeration applied to recombinant protein production by virus-infected insect cells

Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells. The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer. The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture. On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC-medium interface. These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system. Overall mass-transfer coefficient K(L) for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures. The K(L) value in the PFC-mediated system was 2.60x10(-3)cms(-1), 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC-medium and medium-air interfaces, respectively. However, the K(L) values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces. DO behavior in the cultures was well explained by the model calculation using the determined K(L) values and oxygen-consumption rates of viable cells. This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24-0.5ppm (3-7%) in the insect cell/baculovirus expression system.

Correlation of liquid-side mass transfer coefficient based on the new concept of specific power group

Abstract A new concept of a specific power group, proposed previously for correlating the mass transfer between a liquid and particles fixed in the liquid with a low level of turbulence, is discussed in comparison with the conventional concept of a specific power group from the Kolmogoroff theory. The new concept gives a single correlation equation of liquid-side mass transfer coefficients for single particles, fixed beds, and fluidized beds, which is practically the same as the correlation equations for stirred tanks, suspended bubble columns, and two-phase tube flow based on the conventional concept. The agreement induces the consideration of the relationship between specific power groups based on the new concept and the conventional one from the viewpoint of hydrodynamics. The energy dissipation rate for a particle, defined in the new concept, is extended to the particles suspended in a fully developed turbulence field and is hydrodynamically compared with the average energy dissipation rate defined conventionally in the turbulence field. It is revealed that the magnitude of the specific power group including the former energy dissipation rate almost agrees with that including the latter one. Thus, combined with the specific power group based on the conventional concept which has a simplicity in the calculation of its value for suspended particles, the specific power group based on our concept is useful as a general parameter in the study of mass transfer phenomena between a liquid and particles where the liquid is either quiescent or turbulent and the particles are fixed or suspended.

Immobilization of γ-glutamyl transpeptidase, a membrane enzyme, in gel beads via liposome entrapment

Abstract Bovine kidney γ-glutamyl transpeptidase, a membrane enzyme, was immobilized in gel beads by application of the method of Wallsten et al . (Biochim. Biophys. Acta, 982, 47–52, 1989). The gel beads were equilibrated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed against a buffer for reconstitution and immobilization of enzyme-bound liposomes in the pores of the beads. From the standpoints of the immobilized contents of protein and phospholipids and of the reactivity of γ-glutamyl transpeptidase, a dialysis buffer of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in the enzyme-phospholipid-cholate dispersion, and the use of Sepharose CL-6B as the support gel were found to be most appropriate for the immobilization of γ-glutamyl transpeptidase, γ-Glutamyl transpeptidase was activated and stabilized by reconstitution in liposomes. In operation with a packed bed reactor, liposome-bound γ-glutamyl transpeptidase immobilized in Sepharose CL-6B exhibited relatively stable and constant activity for 12 h. In addition, it was found that enzyme substrates were able to pass through the pores of the gel beads to interact with the enzyme present on the outer surface of the liposome membrane in the gel beads. These results thus indicated that a novel support made up of liposomes and Sepharose CL-6B would permit efficient immobilization of lipid-requiring and/or membrane enzymes.

Prorenin Processing Enzyme (PPE) Produced by Baculovirus-Infected Sf-9 Insect Cells: PPE Is the Cysteine Protease Encoded in the AcMNPV Gene

In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610–2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.