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Efficient production of an antibody Fab fragment using the baculovirus–insect cell system
Journal of Bioscience and Bioengineering VOL. 110 No. 5, 577 – 581, 2010 www.elsevier.com/locate/jbiosc
Efficient production of an antibody Fab fragment using the baculovirus–insect cell system Takanori Furuta,1 Takafumi Ogawa,1,2 Tomohisa Katsuda,1 Ikuo Fujii,3 and Hideki Yamaji 1,⁎ Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan 1 Institute of Pathology, Kyodo Byori, Inc., 2-7-12 Otsuwa, Nishiku, Kobe 651-2112, Japan 2 and Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuencho, Nakaku, Sakai, Osaka 599-8531, Japan 3 Received 19 April 2010; accepted 3 June 2010 Available online 29 June 2010
The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (N 600 μg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus–insect cell system may allow efficient production of antibody Fab fragments. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Insect cell; Baculovirus; Recombinant protein production; Antibody; Fab fragment; Signal sequence; gp64]
In recent years the baculovirus–insect cell system has been one of the most widely used systems for the production of biologically active recombinant proteins (1). In this system, a recombinant nucleopolyhedrovirus (NPV) is constructed, in which the nonessential polyhedrin gene is usually replaced with the foreign gene of interest. Subsequent infection of cultured lepidopteran insect cells with the recombinant baculovirus often leads to the expression of extremely large quantities of the foreign protein through higher-eukaryote post-translational processing and modifications under the control of the very strong polyhedrin promoter during the very late stage of infection. The highly restricted host range of the baculovirus makes this expression system safe. A wide variety of recombinant proteins, including both secreted and membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells (1–3). Monoclonal antibodies and antibody fragments, such as Fab and Fab' fragments, have been employed in a variety of diagnostic and therapeutic applications (4,5). These antibody molecules have also been expressed using the baculovirus–insect cell system (6–8). To
⁎ Corresponding author. Tel./fax: +81 78 803 6200. E-mail address: [email protected] (H. Yamaji).
express an intact IgG molecule or an Fab fragment, the genes that encode both the heavy chain (Hc) and light chain (Lc) must be expressed in the same cell. Although expression of both the Hc and Lc genes can be achieved by double infection of insect cells with two recombinant baculoviruses, one containing the Hc gene and the other the Lc gene, this strategy requires the generation of two recombinant baculoviruses and careful adjustment of the infection conditions. Therefore, recombinant baculoviruses that contain both the Hc and Lc cDNAs have been used for the expression of intact IgG (6,8). While much effort has gone into investigating the expression of various antibodies by use of a single recombinant baculovirus, high-level expression based on this strategy has not been examined in detail, except for co-expression of a molecular chaperone (9,10). In the present study, the production of an Fab fragment of the catalytic antibody 6D9 (11) in lepidopteran insect cells infected with a recombinant baculovirus that contained both the Hc and Lc genes was investigated. The effects of various factors on the secretion of 6D9 Fab fragments were examined as follows: promoter and gene combinations, host cells, the culture mode, the multiplicity of infection (MOI), and the signal sequence. The results obtained in the present study provide important information on efficient production of Fab fragments in the baculovirus–insect cell system.
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.06.001
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FURUTA ET AL. MATERIALS AND METHODS
Insect cells and media Spodoptera frugiperda Sf9 (BD Biosciences, San Jose, CA, USA) and Trichoplusia ni BTI-TN-5B1-4 (High Five; Invitrogen, Carlsbad, CA, USA) insect cells were used in the present study. Cell density was determined by microscopically counting the number of cells with a Bürker–Türk hemocytometer, while cell viability was judged by trypan blue dye exclusion (12). The cells were maintained at 27 °C in Tflasks in a non-humidified incubator. TNM-FH was used for routine maintenance of the Sf9 cells, and consisted of 51.19 g/l TNM-FH (Sigma Chemical, St. Louis, MO, USA), 0.35 g/l NaHCO3, 10 mg/l gentamicin sulfate (Invitrogen), and 1 g/l Pluronic F-68 [block co-polymer glycol of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene), Sigma], supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA) (12). TNM-FH is made by supplementing a basal synthetic medium, Grace's medium, with 3.33 g/l lactalbumin hydrolysate and 3.33 g/l yeast extract. Unsupplemented Grace's medium (Invitrogen) was used when Sf9 cells were transfected with recombinant bacmid DNA. Serum-free Express Five medium (Invitrogen) supplemented with 2.41 g/l L-glutamine and 10 mg/l gentamicin was used for the High Five cells throughout. Generation of recombinant baculoviruses The catalytic antibody 6D9, which was generated by immunization of mice with a transition-state analog, catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol (11,13). For the production of Fab fragments of this antibody in the baculovirus– insect cell system, recombinant baculoviruses were constructed using the Bac-to-Bac baculovirus expression system (Invitrogen), which is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in Escherichia coli (14). The cDNA fragments encoding the Hc (Fd fragment) and Lc genes of the Fab fragment with a signal sequence were PCR cloned into pFastBac Dual (Invitrogen), which contained two multiple cloning sites. Thus, one gene was cloned as an NheI–SphI fragment downstream of the Autographa californica NPV (AcNPV) p10 promoter, and the other as an EcoRI–HindIII fragment downstream of the AcNPV polyhedrin promoter. One of the three signal sequences, the Drosophila BiP signal sequence (5'-ATGAAGTTATGCATATTACTGGCCGTCGTGGCCTTTGTTGGCCTCTCGCTCGGG-3') (15), the honeybee melittin signal sequence (5'-ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCC-3') (16), or the AcNPV gp64 signal sequence (5'-ATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCG-3') (17), was included upstream of the Hc and Lc genes. MAX Efficiency DH10Bac competent E. coli cells (Invitrogen) were transformed with the constructed plasmids to generate recombinant bacmids according to the protocol recommended by the manufacturer. The recombinant bacmid DNA was then transfected into Sf9 cells using the Cellfectin reagent (Invitrogen) in unsupplemented Grace's medium, resulting in the release of recombinant baculovirus from transfected cells. High-titer stocks of the recombinant baculovirus were generated from Sf9 cells, the titers of which were determined using the FastPlax titer kit (Merck, Tokyo, Japan). Cell culture and recombinant protein production Cells in the exponential growth phase were collected and suspended at a density of approximately 1× 106 cells/cm3 in fresh medium. Five milliliters of the cell suspension was transferred into T25-flasks, while 15 ml of cell suspension was added to 100-ml screw-capped Erlenmeyer flasks. The hightiter viral stock solution was added to each flask to give a predetermined MOI. The time of viral solution addition was designated as post-infection time zero. The cells in the T-flasks were statically cultivated, and, when appropriate, one of the flasks was removed to obtain a sample of the cell suspension. The cells in the Erlenmeyer flasks were cultivated at 27 °C on a reciprocal shaker (90 oscillations/min; amplitude 25 mm), and aliquots of the cell suspensions were sampled. For both static and shake-flask cultures, cell density was measured, while the cells and culture supernatants were separated via centrifugation. The cell-free supernatants were stored at −20 °C for subsequent measurement of secreted (extracellular) 6D9 Fab fragments. After washing with phosphate-buffered saline (PBS), the cells were lysed in PBS containing 1.0% Triton X-100. Following centrifugation of the cell lysate, the supernatants were stored at −20 °C for subsequent measurement of intracellular 6D9 Fab fragments. Western blot analysis Culture supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12.5% gel under nonreducing conditions, followed by transfer to a poly(vinylidene difluoride) (PVDF) membrane. Western blot analysis was carried out using alkaline phosphatase-conjugated goat anti-mouse IgG (H+ L) (Promega, Madison, WI, USA) and immunoreactive bands were detected using 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (Promega) (15). Enzyme-linked immunosorbent assay The concentration of the 6D9 Fab fragment was measured by enzyme-linked immunosorbent assay (ELISA) using a conjugate of hapten (a transition-state analog for the hydrolysis of a chloramphenicol monoester derivative) and bovine serum albumin (BSA) (13) as the antigen and horseradish peroxidase-conjugated goat anti-mouse IgG, as previously described (15). Immunocytochemistry All subsequent steps were performed at room temperature. Cells were fixed in SurePath preservative (TriPath Imaging, Burlington, NC, USA) 2 days after infection and were collected via centrifugation. A drop of the sediment was placed on a slide glass, and the edge of a second slide glass was used to spread the drop across the first slide glass to make a smear. After the smear had dried, it was immersed in 3% H2O2 for 10 min to inhibit endogenous peroxidase activity. After washing with PBS, the non-specific protein-binding was blocked by treating the smear with a protein-binding agent (Thermo Fisher Scientific, Pittsburgh, PA, USA) for 10 min. After additional washing, the smear was
J. BIOSCI. BIOENG., incubated with horseradish peroxidase-conjugated swine anti-mouse IgG (DAKO North America, Carpinteria, CA, USA) for 30 min. After the final wash, immune complexes were visualized by incubation for 3 min with 0.01% H2O2 and 0.05% 3,3'-diaminobenzidine tetrachloride (DAB) and nuclear counterstaining was accomplished using Mayer's hematoxylin solution for 5 min.
RESULTS AND DISCUSSION Effect of promoter and gene combinations For the production of a recombinant antibody Fab fragment, the genes that encode both the Hc (Fd fragment) and Lc of the Fab fragment must be expressed. In the present study, recombinant baculoviruses were generated using the Bacto-Bac baculovirus expression system (Invitrogen) with the donor plasmid pFastBac Dual, which contains two multiple cloning sites to allow simultaneous expression of two heterologous genes in a single recombinant baculovirus. In the pFastBac Dual, one gene is expressed under the control of the AcNPV polyhedrin promoter, while the other is under the control of the AcNPV p10 promoter. Both the polyhedrin and p10 promoters are strong and “very late” promoters, but reportedly the polyhedrin promoter is generally stronger than the p10 promoter (18,19). Therefore, the promoter (polyhedrin or p10) and gene (Hc or Lc) combination may affect the expression level of the Fab fragment. In the present study, two recombinant baculoviruses, both of which included the AcNPV gp64 signal sequence, were constructed and secretion of 6D9 Fab fragment was compared. The first recombinant baculovirus was p10Hc/PH-Lc, in which the Hc and Lc genes of the 6D9 Fab fragment were located downstream of the AcNPV p10 and polyhedrin (PH) promoters, respectively. The second recombinant baculovirus was p10-Lc/PH-Hc, in which the Lc and Hc genes were downstream of the p10 and polyhedrin promoters, respectively. Fig. 1A shows western blot analysis of the culture supernatants of High Five cells infected with either p10-Hc/PH-Lc or p10-Lc/PH-Hc, both of which employed the AcNPV gp64 signal sequence. In the culture supernatant of the infected cells, specific protein bands were detected at an electrophoretic mobility of approximately 48 kDa, which coincided with the molecular weight of the 6D9 Fab fragment. The western blot analysis also indicated that secretion of the 6D9 Fab fragment by High Five cells was greater with p10-Lc/PH-Hc compared to p10-Hc/PH-Lc. Culture supernatants of High Five cells infected with the recombinant baculoviruses were also analyzed by ELISA (Fig. 1B). Whereas a strong signal was obtained for the culture supernatants of cells infected with either of the two recombinant baculoviruses, the secretion of the 6D9 Fab fragment by cells infected with p10-Lc/PHHc was increased approximately two-fold compared with that obtained with p10-Hc/PH-Lc. A similar result was obtained using Sf9 cells infected with either p10-Lc/PH-Hc or p10-Hc/PH-Lc, both of which contained the Drosophila BiP signal sequence (data not shown). Immunocytochemical staining of High Five cells infected with the recombinant baculovirus p10-Lc/PH-Hc using the gp64 signal sequence 2 days after infection suggested that the Fab fragment was actually expressed in the cytoplasm outside the cell nucleus, which had increased in size (Fig. 2). These results suggest that 6D9 Fab fragment that is capable of binding the corresponding antigen was secreted by baculovirus-infected insect cells into the culture medium. Due to the high-expression level, the recombinant baculovirus p10Lc/PH-Hc, in which the Lc and Hc genes were expressed under the control of the p10 and polyhedrin promoters, respectively, was used in subsequent investigations. Effects of host cells, culture mode, and MOI Lepidopteran Sf9 and High Five cells are commonly used as host cells for infection with recombinant AcNPV in the baculovirus–insect cell system. High Five cells have been shown to be superior to Sf9 cells for expression of secreted recombinant proteins (20–22). However, this does not hold true for the expression of all proteins (23–25). Upon infection with the recombinant baculovirus p10-Lc/PH-Hc that included the AcNPV
VOL. 110, 2010 gp64 signal sequence, High Five cells produced a greater 6D9 Fab fragment yield than Sf9 cells in the culture medium in both static and shake-flask cultures (Fig. 3). Because both types of cells were infected at the same cell density and at the same MOI, the specific productivity of High Five cells was higher than that of Sf9 cells, though the culture medium used for High Five cells was different from that for Sf9 cells. For both High Five and Sf9 cells, the yields of the 6D9 Fab fragment in the shake-flask cultures were increased nearly two-fold compared with the static cultures. The increased Fab fragment yields were probably due to better oxygen supply in the shake-flask cultures. Thus, High Five cells and shake-flask culture were selected for use in subsequent investigations. Fig. 4 shows the time course of viable cell density and secretory Fab production in shake-flask cultures infected at MOIs of 10, 1, and 0.1 plaque-forming units (pfu)/cell. On infection at a low MOI (i.e., 0.1 pfu/cell), the decrease in the viable cell density and the increase in the Fab fragment yield were delayed as compared with highmultiplicity infections, since only a small proportion of cells had been initially infected. In the cultures infected at MOIs of 1 or 10 pfu/cell, nearly identical changes in viable cell density and Fab fragment concentration were observed, because essentially all cells were infected immediately after addition of the virus stock solution. These results were highly consistent with previous observations (12). Comparison of signal sequence Next, the effect of signal sequence on the secretion of the 6D9 Fab fragment by baculovirusinfected High Five cells was investigated. Fig. 5 shows the time course of Fab fragment production by the cells infected with recombinant baculovirus that differed in the signal sequence upstream of the Hc and Lc genes. Interestingly, the signal sequence markedly affected the secretion of the Fab fragment into the culture medium. A higher yield of the Fab fragment (greater than 600 μg/ml) was obtained with the baculovirus gp64 signal sequence compared with the other insectderived signal sequences. gp64 is the major envelope surface glycoprotein of AcNPV and plays an important role in infection (17). During the infection cycle, gp64 is abundantly expressed and transported to the surface of infected cells for incorporation into budding virions. It is possible that a baculovirus signal sequence is
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FIG. 2. Immunocytochemical staining of High Five cells infected with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence. Cells were infected at an MOI of 10 pfu/cell and harvested 2 days after infection. Fab fragments were stained brown using horseradish peroxidase-conjugated anti-mouse IgG and the nucleus was stained blue with hematoxylin.
well suited for secretion in the baculovirus–insect cell system (see below). Fig. 5 also shows the time course of total (extracellular and intracellular) Fab fragment production by baculovirus-infected High Five cells. The proportions of secreted Fab fragment were calculated and found to be approximately 58% during 2–4 days. There were no notable differences among the signal sequences tested in the present study, indicating that signal sequences may affect the production level but not the secretion efficiency. Whereas the secretion of the Fab fragment using each signal sequence reached a plateau on 4 days, the total Fab fragment concentration increased up to 5 days, suggesting that the host cell secretory pathway was compromised during 4– 5 days after infection.
FIG. 1. Effect of promoter and gene combinations on secretion of 6D9 Fab fragments by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells infected with recombinant baculovirus. (A) Western blot analysis of culture supernatant. (B) Enzyme-linked immunosorbent assay (ELISA) of cell culture supernatant. Cells were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell and incubated for 3 days in static culture. The recombinant baculovirus p10-Hc/PH-Lc included the Hc and Lc genes of the Fab fragment with the Autographa californica nucleopolyhedrovirus (AcNPV) gp64 signal sequence downstream of the AcNPV p10 and polyhedrin (PH) promoters, respectively; p10-Lc/PH-Hc included the Lc and Hc genes downstream of the p10 and polyhedrin promoters, respectively.
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FIG. 3. Effect of host insect cells on production of 6D9 Fab fragment in static and shakeflask cultures. Spodoptera frugiperda Sf9 and High Five cells were infected at an MOI of 10 pfu/cell with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence. After 3 days of incubation, the culture supernatant was analyzed by ELISA.
FIG. 4. Effect of MOI on 6D9 Fab fragment production by baculovirus-infected High Five cells in shake-flask culture. Cells were infected at an MOI of 10 (circles), 1 (squares), or 0.1 (triangles) pfu/cell with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence.
J. BIOSCI. BIOENG., Reportedly, the expression and secretion of HIV-1 gp120 in baculovirus-infected Sf9 cells were improved when the signal sequence of baculovirus ecdysteroid UDPglucosyltransferase or gp67 (syn.: gp64) was used (26). On the other hand, the use of a signal sequence from lepidopteran insect cecropin B, honeybee melittin, or gp64 had no significant effect on the expression and secretion of human tissue plasminogen activator in baculovirus-infected Sf9 cells (27). No significant difference in the production or secretion efficiency of β1, 3-N-acetylglucosaminyltransferase 2 fused with green fluorescence protein in baculovirus-infected High Five cells was observed by use of the signal sequences from melittin, gp64, or cecropin B (28). These reports suggest that inclusion of a baculovirus signal sequence does not always enhance the production of a recombinant secreted protein in the baculovirus–insect cell system and that the resultant production level might depend on the protein to be expressed. However, our results indicate that the AcNPV gp64 signal sequence might be a viable choice for the production of recombinant antibody molecules. In a previous study, we investigated the production of the 6D9 Fab fragment in stably transformed High Five cells (15). After cotransfection with the Hc and Lc genes of the 6D9 Fab fragment, which were cloned separately into two plasmid vectors that each contained the baculoviral IE-1 transactivator, the baculoviral HR3 enhancer, and the Bombyx mori actin promoter, and either a neomycin- or a blasticidin-resistance gene, respectively, High Five cells stably secreting a high concentration of the functional Fab fragment were successfully generated by incubation in the presence of G418 and blasticidin for 30 days. A high Fab fragment yield of approximately 350 μg/ml was achieved in a shake-flask culture of the recombinant insect cells after 6 days (15). Although a simple comparison of the results of recombinant insect cells with those of baculovirus-infected insect cells is impossible, higher yields of the secreted 6D9 Fab fragment were obtained by baculovirus-infected High Five cells in shake-flask culture for 5 days in the present study (Figs. 4 and 5). Recently, Fab and Fab' fragments have been used as therapeutics (4,5). Although continuous protein production is virtually impossible in the baculovirus–insect cell system because of the lytic nature of the viral infection process, the results obtained in the present study indicate that this system may provide a rapid and simple way of producing large amounts of recombinant Fab fragments.
FIG. 5. Effect of signal sequence on 6D9 Fab fragment production by baculovirus-infected High Five cells in shake-flask culture. Cells were infected at an MOI of 10 pfu/cell with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence (circles), the Drosophila BiP signal sequence (squares), or the honeybee melittin signal sequence (triangles). Open symbols, secreted (extracellular) Fab fragment concentration; closed symbols, total (intracellular and extracellular) Fab fragment concentration.
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FAB PRODUCTION IN THE BACULOVIRUS-INSECT CELL SYSTEM ACKNOWLEDGMENTS
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Efficient production of an antibody Fab fragment using the baculovirus–insect cell system Takanori Furuta,1 Takafumi Ogawa,1,2 Tomohisa Katsuda,1 Ikuo Fujii,3 and Hideki Yamaji 1,⁎ Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan 1 Institute of Pathology, Kyodo Byori, Inc., 2-7-12 Otsuwa, Nishiku, Kobe 651-2112, Japan 2 and Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuencho, Nakaku, Sakai, Osaka 599-8531, Japan 3 Received 19 April 2010; accepted 3 June 2010 Available online 29 June 2010
The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (N 600 μg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus–insect cell system may allow efficient production of antibody Fab fragments. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Insect cell; Baculovirus; Recombinant protein production; Antibody; Fab fragment; Signal sequence; gp64]
In recent years the baculovirus–insect cell system has been one of the most widely used systems for the production of biologically active recombinant proteins (1). In this system, a recombinant nucleopolyhedrovirus (NPV) is constructed, in which the nonessential polyhedrin gene is usually replaced with the foreign gene of interest. Subsequent infection of cultured lepidopteran insect cells with the recombinant baculovirus often leads to the expression of extremely large quantities of the foreign protein through higher-eukaryote post-translational processing and modifications under the control of the very strong polyhedrin promoter during the very late stage of infection. The highly restricted host range of the baculovirus makes this expression system safe. A wide variety of recombinant proteins, including both secreted and membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells (1–3). Monoclonal antibodies and antibody fragments, such as Fab and Fab' fragments, have been employed in a variety of diagnostic and therapeutic applications (4,5). These antibody molecules have also been expressed using the baculovirus–insect cell system (6–8). To
⁎ Corresponding author. Tel./fax: +81 78 803 6200. E-mail address: [email protected] (H. Yamaji).
express an intact IgG molecule or an Fab fragment, the genes that encode both the heavy chain (Hc) and light chain (Lc) must be expressed in the same cell. Although expression of both the Hc and Lc genes can be achieved by double infection of insect cells with two recombinant baculoviruses, one containing the Hc gene and the other the Lc gene, this strategy requires the generation of two recombinant baculoviruses and careful adjustment of the infection conditions. Therefore, recombinant baculoviruses that contain both the Hc and Lc cDNAs have been used for the expression of intact IgG (6,8). While much effort has gone into investigating the expression of various antibodies by use of a single recombinant baculovirus, high-level expression based on this strategy has not been examined in detail, except for co-expression of a molecular chaperone (9,10). In the present study, the production of an Fab fragment of the catalytic antibody 6D9 (11) in lepidopteran insect cells infected with a recombinant baculovirus that contained both the Hc and Lc genes was investigated. The effects of various factors on the secretion of 6D9 Fab fragments were examined as follows: promoter and gene combinations, host cells, the culture mode, the multiplicity of infection (MOI), and the signal sequence. The results obtained in the present study provide important information on efficient production of Fab fragments in the baculovirus–insect cell system.
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.06.001
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Insect cells and media Spodoptera frugiperda Sf9 (BD Biosciences, San Jose, CA, USA) and Trichoplusia ni BTI-TN-5B1-4 (High Five; Invitrogen, Carlsbad, CA, USA) insect cells were used in the present study. Cell density was determined by microscopically counting the number of cells with a Bürker–Türk hemocytometer, while cell viability was judged by trypan blue dye exclusion (12). The cells were maintained at 27 °C in Tflasks in a non-humidified incubator. TNM-FH was used for routine maintenance of the Sf9 cells, and consisted of 51.19 g/l TNM-FH (Sigma Chemical, St. Louis, MO, USA), 0.35 g/l NaHCO3, 10 mg/l gentamicin sulfate (Invitrogen), and 1 g/l Pluronic F-68 [block co-polymer glycol of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene), Sigma], supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA) (12). TNM-FH is made by supplementing a basal synthetic medium, Grace's medium, with 3.33 g/l lactalbumin hydrolysate and 3.33 g/l yeast extract. Unsupplemented Grace's medium (Invitrogen) was used when Sf9 cells were transfected with recombinant bacmid DNA. Serum-free Express Five medium (Invitrogen) supplemented with 2.41 g/l L-glutamine and 10 mg/l gentamicin was used for the High Five cells throughout. Generation of recombinant baculoviruses The catalytic antibody 6D9, which was generated by immunization of mice with a transition-state analog, catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol (11,13). For the production of Fab fragments of this antibody in the baculovirus– insect cell system, recombinant baculoviruses were constructed using the Bac-to-Bac baculovirus expression system (Invitrogen), which is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in Escherichia coli (14). The cDNA fragments encoding the Hc (Fd fragment) and Lc genes of the Fab fragment with a signal sequence were PCR cloned into pFastBac Dual (Invitrogen), which contained two multiple cloning sites. Thus, one gene was cloned as an NheI–SphI fragment downstream of the Autographa californica NPV (AcNPV) p10 promoter, and the other as an EcoRI–HindIII fragment downstream of the AcNPV polyhedrin promoter. One of the three signal sequences, the Drosophila BiP signal sequence (5'-ATGAAGTTATGCATATTACTGGCCGTCGTGGCCTTTGTTGGCCTCTCGCTCGGG-3') (15), the honeybee melittin signal sequence (5'-ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCC-3') (16), or the AcNPV gp64 signal sequence (5'-ATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCG-3') (17), was included upstream of the Hc and Lc genes. MAX Efficiency DH10Bac competent E. coli cells (Invitrogen) were transformed with the constructed plasmids to generate recombinant bacmids according to the protocol recommended by the manufacturer. The recombinant bacmid DNA was then transfected into Sf9 cells using the Cellfectin reagent (Invitrogen) in unsupplemented Grace's medium, resulting in the release of recombinant baculovirus from transfected cells. High-titer stocks of the recombinant baculovirus were generated from Sf9 cells, the titers of which were determined using the FastPlax titer kit (Merck, Tokyo, Japan). Cell culture and recombinant protein production Cells in the exponential growth phase were collected and suspended at a density of approximately 1× 106 cells/cm3 in fresh medium. Five milliliters of the cell suspension was transferred into T25-flasks, while 15 ml of cell suspension was added to 100-ml screw-capped Erlenmeyer flasks. The hightiter viral stock solution was added to each flask to give a predetermined MOI. The time of viral solution addition was designated as post-infection time zero. The cells in the T-flasks were statically cultivated, and, when appropriate, one of the flasks was removed to obtain a sample of the cell suspension. The cells in the Erlenmeyer flasks were cultivated at 27 °C on a reciprocal shaker (90 oscillations/min; amplitude 25 mm), and aliquots of the cell suspensions were sampled. For both static and shake-flask cultures, cell density was measured, while the cells and culture supernatants were separated via centrifugation. The cell-free supernatants were stored at −20 °C for subsequent measurement of secreted (extracellular) 6D9 Fab fragments. After washing with phosphate-buffered saline (PBS), the cells were lysed in PBS containing 1.0% Triton X-100. Following centrifugation of the cell lysate, the supernatants were stored at −20 °C for subsequent measurement of intracellular 6D9 Fab fragments. Western blot analysis Culture supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12.5% gel under nonreducing conditions, followed by transfer to a poly(vinylidene difluoride) (PVDF) membrane. Western blot analysis was carried out using alkaline phosphatase-conjugated goat anti-mouse IgG (H+ L) (Promega, Madison, WI, USA) and immunoreactive bands were detected using 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (Promega) (15). Enzyme-linked immunosorbent assay The concentration of the 6D9 Fab fragment was measured by enzyme-linked immunosorbent assay (ELISA) using a conjugate of hapten (a transition-state analog for the hydrolysis of a chloramphenicol monoester derivative) and bovine serum albumin (BSA) (13) as the antigen and horseradish peroxidase-conjugated goat anti-mouse IgG, as previously described (15). Immunocytochemistry All subsequent steps were performed at room temperature. Cells were fixed in SurePath preservative (TriPath Imaging, Burlington, NC, USA) 2 days after infection and were collected via centrifugation. A drop of the sediment was placed on a slide glass, and the edge of a second slide glass was used to spread the drop across the first slide glass to make a smear. After the smear had dried, it was immersed in 3% H2O2 for 10 min to inhibit endogenous peroxidase activity. After washing with PBS, the non-specific protein-binding was blocked by treating the smear with a protein-binding agent (Thermo Fisher Scientific, Pittsburgh, PA, USA) for 10 min. After additional washing, the smear was
J. BIOSCI. BIOENG., incubated with horseradish peroxidase-conjugated swine anti-mouse IgG (DAKO North America, Carpinteria, CA, USA) for 30 min. After the final wash, immune complexes were visualized by incubation for 3 min with 0.01% H2O2 and 0.05% 3,3'-diaminobenzidine tetrachloride (DAB) and nuclear counterstaining was accomplished using Mayer's hematoxylin solution for 5 min.
RESULTS AND DISCUSSION Effect of promoter and gene combinations For the production of a recombinant antibody Fab fragment, the genes that encode both the Hc (Fd fragment) and Lc of the Fab fragment must be expressed. In the present study, recombinant baculoviruses were generated using the Bacto-Bac baculovirus expression system (Invitrogen) with the donor plasmid pFastBac Dual, which contains two multiple cloning sites to allow simultaneous expression of two heterologous genes in a single recombinant baculovirus. In the pFastBac Dual, one gene is expressed under the control of the AcNPV polyhedrin promoter, while the other is under the control of the AcNPV p10 promoter. Both the polyhedrin and p10 promoters are strong and “very late” promoters, but reportedly the polyhedrin promoter is generally stronger than the p10 promoter (18,19). Therefore, the promoter (polyhedrin or p10) and gene (Hc or Lc) combination may affect the expression level of the Fab fragment. In the present study, two recombinant baculoviruses, both of which included the AcNPV gp64 signal sequence, were constructed and secretion of 6D9 Fab fragment was compared. The first recombinant baculovirus was p10Hc/PH-Lc, in which the Hc and Lc genes of the 6D9 Fab fragment were located downstream of the AcNPV p10 and polyhedrin (PH) promoters, respectively. The second recombinant baculovirus was p10-Lc/PH-Hc, in which the Lc and Hc genes were downstream of the p10 and polyhedrin promoters, respectively. Fig. 1A shows western blot analysis of the culture supernatants of High Five cells infected with either p10-Hc/PH-Lc or p10-Lc/PH-Hc, both of which employed the AcNPV gp64 signal sequence. In the culture supernatant of the infected cells, specific protein bands were detected at an electrophoretic mobility of approximately 48 kDa, which coincided with the molecular weight of the 6D9 Fab fragment. The western blot analysis also indicated that secretion of the 6D9 Fab fragment by High Five cells was greater with p10-Lc/PH-Hc compared to p10-Hc/PH-Lc. Culture supernatants of High Five cells infected with the recombinant baculoviruses were also analyzed by ELISA (Fig. 1B). Whereas a strong signal was obtained for the culture supernatants of cells infected with either of the two recombinant baculoviruses, the secretion of the 6D9 Fab fragment by cells infected with p10-Lc/PHHc was increased approximately two-fold compared with that obtained with p10-Hc/PH-Lc. A similar result was obtained using Sf9 cells infected with either p10-Lc/PH-Hc or p10-Hc/PH-Lc, both of which contained the Drosophila BiP signal sequence (data not shown). Immunocytochemical staining of High Five cells infected with the recombinant baculovirus p10-Lc/PH-Hc using the gp64 signal sequence 2 days after infection suggested that the Fab fragment was actually expressed in the cytoplasm outside the cell nucleus, which had increased in size (Fig. 2). These results suggest that 6D9 Fab fragment that is capable of binding the corresponding antigen was secreted by baculovirus-infected insect cells into the culture medium. Due to the high-expression level, the recombinant baculovirus p10Lc/PH-Hc, in which the Lc and Hc genes were expressed under the control of the p10 and polyhedrin promoters, respectively, was used in subsequent investigations. Effects of host cells, culture mode, and MOI Lepidopteran Sf9 and High Five cells are commonly used as host cells for infection with recombinant AcNPV in the baculovirus–insect cell system. High Five cells have been shown to be superior to Sf9 cells for expression of secreted recombinant proteins (20–22). However, this does not hold true for the expression of all proteins (23–25). Upon infection with the recombinant baculovirus p10-Lc/PH-Hc that included the AcNPV
VOL. 110, 2010 gp64 signal sequence, High Five cells produced a greater 6D9 Fab fragment yield than Sf9 cells in the culture medium in both static and shake-flask cultures (Fig. 3). Because both types of cells were infected at the same cell density and at the same MOI, the specific productivity of High Five cells was higher than that of Sf9 cells, though the culture medium used for High Five cells was different from that for Sf9 cells. For both High Five and Sf9 cells, the yields of the 6D9 Fab fragment in the shake-flask cultures were increased nearly two-fold compared with the static cultures. The increased Fab fragment yields were probably due to better oxygen supply in the shake-flask cultures. Thus, High Five cells and shake-flask culture were selected for use in subsequent investigations. Fig. 4 shows the time course of viable cell density and secretory Fab production in shake-flask cultures infected at MOIs of 10, 1, and 0.1 plaque-forming units (pfu)/cell. On infection at a low MOI (i.e., 0.1 pfu/cell), the decrease in the viable cell density and the increase in the Fab fragment yield were delayed as compared with highmultiplicity infections, since only a small proportion of cells had been initially infected. In the cultures infected at MOIs of 1 or 10 pfu/cell, nearly identical changes in viable cell density and Fab fragment concentration were observed, because essentially all cells were infected immediately after addition of the virus stock solution. These results were highly consistent with previous observations (12). Comparison of signal sequence Next, the effect of signal sequence on the secretion of the 6D9 Fab fragment by baculovirusinfected High Five cells was investigated. Fig. 5 shows the time course of Fab fragment production by the cells infected with recombinant baculovirus that differed in the signal sequence upstream of the Hc and Lc genes. Interestingly, the signal sequence markedly affected the secretion of the Fab fragment into the culture medium. A higher yield of the Fab fragment (greater than 600 μg/ml) was obtained with the baculovirus gp64 signal sequence compared with the other insectderived signal sequences. gp64 is the major envelope surface glycoprotein of AcNPV and plays an important role in infection (17). During the infection cycle, gp64 is abundantly expressed and transported to the surface of infected cells for incorporation into budding virions. It is possible that a baculovirus signal sequence is
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FIG. 2. Immunocytochemical staining of High Five cells infected with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence. Cells were infected at an MOI of 10 pfu/cell and harvested 2 days after infection. Fab fragments were stained brown using horseradish peroxidase-conjugated anti-mouse IgG and the nucleus was stained blue with hematoxylin.
well suited for secretion in the baculovirus–insect cell system (see below). Fig. 5 also shows the time course of total (extracellular and intracellular) Fab fragment production by baculovirus-infected High Five cells. The proportions of secreted Fab fragment were calculated and found to be approximately 58% during 2–4 days. There were no notable differences among the signal sequences tested in the present study, indicating that signal sequences may affect the production level but not the secretion efficiency. Whereas the secretion of the Fab fragment using each signal sequence reached a plateau on 4 days, the total Fab fragment concentration increased up to 5 days, suggesting that the host cell secretory pathway was compromised during 4– 5 days after infection.
FIG. 1. Effect of promoter and gene combinations on secretion of 6D9 Fab fragments by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells infected with recombinant baculovirus. (A) Western blot analysis of culture supernatant. (B) Enzyme-linked immunosorbent assay (ELISA) of cell culture supernatant. Cells were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell and incubated for 3 days in static culture. The recombinant baculovirus p10-Hc/PH-Lc included the Hc and Lc genes of the Fab fragment with the Autographa californica nucleopolyhedrovirus (AcNPV) gp64 signal sequence downstream of the AcNPV p10 and polyhedrin (PH) promoters, respectively; p10-Lc/PH-Hc included the Lc and Hc genes downstream of the p10 and polyhedrin promoters, respectively.
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FIG. 3. Effect of host insect cells on production of 6D9 Fab fragment in static and shakeflask cultures. Spodoptera frugiperda Sf9 and High Five cells were infected at an MOI of 10 pfu/cell with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence. After 3 days of incubation, the culture supernatant was analyzed by ELISA.
FIG. 4. Effect of MOI on 6D9 Fab fragment production by baculovirus-infected High Five cells in shake-flask culture. Cells were infected at an MOI of 10 (circles), 1 (squares), or 0.1 (triangles) pfu/cell with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence.
J. BIOSCI. BIOENG., Reportedly, the expression and secretion of HIV-1 gp120 in baculovirus-infected Sf9 cells were improved when the signal sequence of baculovirus ecdysteroid UDPglucosyltransferase or gp67 (syn.: gp64) was used (26). On the other hand, the use of a signal sequence from lepidopteran insect cecropin B, honeybee melittin, or gp64 had no significant effect on the expression and secretion of human tissue plasminogen activator in baculovirus-infected Sf9 cells (27). No significant difference in the production or secretion efficiency of β1, 3-N-acetylglucosaminyltransferase 2 fused with green fluorescence protein in baculovirus-infected High Five cells was observed by use of the signal sequences from melittin, gp64, or cecropin B (28). These reports suggest that inclusion of a baculovirus signal sequence does not always enhance the production of a recombinant secreted protein in the baculovirus–insect cell system and that the resultant production level might depend on the protein to be expressed. However, our results indicate that the AcNPV gp64 signal sequence might be a viable choice for the production of recombinant antibody molecules. In a previous study, we investigated the production of the 6D9 Fab fragment in stably transformed High Five cells (15). After cotransfection with the Hc and Lc genes of the 6D9 Fab fragment, which were cloned separately into two plasmid vectors that each contained the baculoviral IE-1 transactivator, the baculoviral HR3 enhancer, and the Bombyx mori actin promoter, and either a neomycin- or a blasticidin-resistance gene, respectively, High Five cells stably secreting a high concentration of the functional Fab fragment were successfully generated by incubation in the presence of G418 and blasticidin for 30 days. A high Fab fragment yield of approximately 350 μg/ml was achieved in a shake-flask culture of the recombinant insect cells after 6 days (15). Although a simple comparison of the results of recombinant insect cells with those of baculovirus-infected insect cells is impossible, higher yields of the secreted 6D9 Fab fragment were obtained by baculovirus-infected High Five cells in shake-flask culture for 5 days in the present study (Figs. 4 and 5). Recently, Fab and Fab' fragments have been used as therapeutics (4,5). Although continuous protein production is virtually impossible in the baculovirus–insect cell system because of the lytic nature of the viral infection process, the results obtained in the present study indicate that this system may provide a rapid and simple way of producing large amounts of recombinant Fab fragments.
FIG. 5. Effect of signal sequence on 6D9 Fab fragment production by baculovirus-infected High Five cells in shake-flask culture. Cells were infected at an MOI of 10 pfu/cell with the recombinant baculovirus p10-Lc/PH-Hc that contained the AcNPV gp64 signal sequence (circles), the Drosophila BiP signal sequence (squares), or the honeybee melittin signal sequence (triangles). Open symbols, secreted (extracellular) Fab fragment concentration; closed symbols, total (intracellular and extracellular) Fab fragment concentration.
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FAB PRODUCTION IN THE BACULOVIRUS-INSECT CELL SYSTEM ACKNOWLEDGMENTS
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