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Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter
Biochemical Engineering Journal 24 (2005) 27–30
Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter Nimish G. Dalal a , William E. Bentley a , Hyung Joon Cha b, ∗ b
a Center for Biosystems Research and Department of Chemical Engineering, University of Maryland, College Park, MD 20742, USA Department of Chemical Engineering and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea
Received 3 September 2004; received in revised form 17 January 2005; accepted 18 January 2005
Abstract A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) was constructed so that the green fluorescent protein (GFP) was produced via the early-to-late (ETL) promoter. This enabled rapid monitoring of the infection of Sf-9 insect cells. Notably, GFP and its fluorescence appeared ∼18 h prior to proteins expressed using very late polyhedrin (Polh) promoter. It is anticipated that the use of GFP under the control of ETL promoter will facilitate vector construction, virus isolation, and titer determination. © 2005 Elsevier B.V. All rights reserved. Keywords: Baculovirus infection; Early ETL promoter; Green fluorescent protein; Recombinant protein production
1. Introduction The baculovirus expression vector (BEV) system is both effective and convenient for the overproduction of recombinant proteins in eukaryotic cells [1–5]. The strong polyhedrin (Polh) promoter [6,7] and the 10 kDa fibrous polypeptide (P10) promoter [3,8], are both active in the very late phase of virus infection and when used to drive heterologous protein production can result in the accumulation of over 50% of the total protein [9]. The use of these promoters to establish the progress of infection and protein production would require waiting as long as 48 h post infection. Crawford and Miller [10] characterized the role of several early baculovirus genes on the expression of late viral genes. They also noted the -galactosidase produced under the early-to-late (ETL) promoter could be useful in aiding the identification of occlusionbody negative recombinants. Visualization of the infection process can be particularly difficult. The availability of a reporter protein can greatly facilitate developmental work ∗
Corresponding author. Tel.: +82 54 2792280; fax: +82 54 2795528. E-mail address: [email protected] (H.J. Cha).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.01.026
[11–13]. Richardson et al. developed a method, which uses the -galactosidase under control of early and late promoters including the ETL promoter to help aid in plaque assays [11]. Alternative techniques for virus titration continue to emerge [14]. In the previous work, we employed green fluorescent protein (GFP) as a monitoring reporter for a simple and rapid visualization of baculovirus, AcMNPV, infection [15,16]. In the present work, we have coupled, for the first time, the advantages of ETL promoter with GFP for a more simple and early monitoring of baculovirus infection.
2. Materials and methods 2.1. Construction of expression vector Several baculoviruses were constructed for this work. First, a pBlueBacHis2(lacZ) transfer vector was constructed by polymerase chain reaction (PCR) amplification around the lacZ gene sequence of the pBlueBacHis2/CAT transfer vector (Invitrogen, USA). The gfpuv gene was ex-
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N.G. Dalal et al. / Biochemical Engineering Journal 24 (2005) 27–30
Fig. 1. Gene map of recombinant transfer vector pBBH(lacZ)GFPuv and pBBH-GFPuv/CAT. Abbreviation: PPH , polyhedrin promoter; PETL , early-to-late promoter; ColE1, Escherichia coli replication origin; Amp, ampicillin resistant gene; RS, recombination site; GFPuv, UV-optimized gfp gene; CAT, cat gene; (His)6 , histidine affinity ligand; EK, enterokinase cleavage site; lacZ, -galactosidase gene.
cised from the pGFPuv plasmid (Clontech, USA) using EagI and XbaI (Promega, USA) digestion and the gfpuv gene was inserted in frame under the ETL expression promoter resulting in a transfer vector denoted pBBH(lacZ)GFPuv (Fig. 1A). Then, a recombinant baculovirus, vPETL -GFPuv, was obtained by co-transfection of pBBH(lacZ)GFPuv with wild type AcNPV DNA (Bac-N-BlueTM baculovirus DNA, Invitrogen) into Sf-9 insect cells [17]. Successful cotransfection was observed through green fluorescence using a fluorescence microscope and virus amplification and titer were performed as described previously [15]. A pBBHGFPuv/CAT fusion transfer vector (Fig. 1B) was constructed from pBlueBacHis2-CAT (Invitrogen), containing the Polh promoter for expression of fusion foreign protein, GFP and chloramphenicol acetyl-transferase (CAT) [18,19]. Recombinant virus vPH-GFPuv/CAT was obtained and titered in a similar manner as vPETL -GFPuv. 2.2. Cell culture and sample preparation Sf-9 insect cells (ATCC, CRL-1711) were grown in SF900-II SFM (Invitrogen) and subcultured routinely every 3–4 days [4]. Experiments were performed on 230 ml cell cultures (250 ml spinner flasks) divided from single inocula grown at 27 ◦ C. Infection with the baculoviruses was performed during exponential growth (∼1 × 106 cells ml−1 ) with a predetermined volume of virus stock solution to yield a multiplicity of infection (MOI) of 5, at time denoted t = 0. Culture samples were collected initially every 6 h and then every 12 h after 60 h post infection (hpi). 2.3. Analytical methods Total cell counts were performed using a double chamber hemacytometer (Fisher Scientific, USA), and viability was determined by a Trypan blue (Sigma, USA) exclusion assay using a 0.4% solution. GFP was quantified using whole cell fraction without medium by fluorescence spectrometry
(MPF-66; Perkin-Elmer, USA) at an excitation wavelength of 395 nm and an emission wavelength of 509 nm. Also, GFP from whole cell fraction was assayed using Western blots. 2.4. Western blot analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by mixing a sample with sample buffer (0.5 M Tris–HCl, pH 6.8, 10% glycerol, 5% SDS, 5% -mercaptoethanol, and 0.25% bromophenol blue), incubating at 100 ◦ C for 3 min, centrifuging for 1 min, and loading onto a 15% slab gel. After electrophoresis, the gel was transferred onto a nitrocellulose membrane (Bio-Rad, USA) with a Bio-Rad Mini-Trans Blot Cell in Bjerrum and Schafer-Nielsen transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol; pH 9.2) for 20 min at 10 V followed by 20 min at 20 V. The nitrocellulose membrane was probed with 1:2000 dilution of polyclonal anti-rGFP antibody (Clontech), and detected with 1:5000 dilution of goat anti-rabbit IgG conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, USA) and BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) color development reagent (Sigma).
3. Results and discussion Suspended cell cultures (230 ml) were infected and monitored over the course of infection. Effectiveness of baculovirus infection depends on cell culture conditions such as virus titer, initial cell number, initial cell viability, and medium composition. It was already shown that there is a correlation between baculovirus infection efficiency and GFP production [15,19]. Uninfected cultures doubled in population number, increasing to a maximum of 2.6 × 106 cells ml−1 (data not shown). Cell densities for the infected cultures remained relatively constant post infection, near 1.5 × 106 cells ml−1 (data not shown). The viabilities for
N.G. Dalal et al. / Biochemical Engineering Journal 24 (2005) 27–30
29
Fig. 2. Western blot analyses of GFP expression under ETL () and Polh () promoters, (A) developed Western blots and (B) quantified results that were normalized to the first non-zero intensities from scanned images (12 hpi, ETL signal).
the infected cultures declined rapidly after 60 hpi, while the uninfected culture declined gradually to approximately 70% viability by the end of the cultures (data not shown). A time course for GFP production was examined via Western blot (Fig. 2), indicating a clear shift of ∼18 h for ETLdriven expression in comparison to that expressed via the Polh promoter. The ETL-driven GFP reached a maximum at 50 hpi, after which the accumulation level declined steadily. The decrease was possibly due to proteolysis [20] and/or cell lysis following virus infection, although the latter is less likely given the previously observed persistence of GFP from the very late Polh vectors. Under the Polh promoter, GFP was detected at approximately 42 hpi with a maximum at 96 hpi followed by a sharp decrease. These results correspond well to the expected profile, where immediate-early and delayedearly genes are expressed between 0 and 10 hpi, the transition to the late phase occurring between 10 and 15 hpi, and the very late genes expressed after 18 hpi [21]. Fig. 3A depicts the GFP fluorescence intensities over time. Consistent with Western blots, the GFP fluorescence intensity under the Polh promoter increased rapidly 42 hpi until reaching a maximum at ∼96 hpi, but without a marked decline. As the Western blots indicated a decrease from 96 to 108 h while fluorescence increased, we expect the loss of GFP on Western blot was most likely due to partial degradation without loss of the chromophore, as opposed to lysis, which would additionally diminish fluorescence. This is consistent with our previous study [16]. GFP has chromophore structure that is responsible for its fluorescence and protected by outside barrel (or -can) structure [22]. It was known that GFP could reveal fluorescence unless chromophore structure is not ruined even though full length of protein will be reduced due to -barrel structure breakage by proteolysis [23,24]. Under the early ETL promoter, GFP fluorescence remained low and fairly constant after 48 hpi, but increased initially near 18 hpi
Fig. 3. GFP fluorescence intensity for control (䊉) and cultures infected with vPETL -GFPuv () and vPH-GFPuv/CAT (), (A) fluorescence intensity over entire culture time and (B) fluorescence intensity for first 48 hpi.
(see insert Fig. 3B) resulting in a similar 18 h time shift observed by Western blot analysis.
4. Conclusions GFP production under the ETL promoter showed a clear shift of ∼18 h in comparison to that under the very late Polh promoter. Importantly, these results show that ∼18 h earlier expression of GFP under ETL promoter can facilitate more rapid monitoring in progress of recombinant baculovirus infection for application to virus construction, virus isolation, and titer determination.
References [1] L.K. Miller, Baculoviruses as gene expression vectors, Annu. Rev. Microbiol. 42 (1988) 177–199. [2] V.A. Luckow, M.D. Summers, Trends in the development of baculovirus expression vectors, Biotechnology 6 (1988) 47–55. [3] L.A. King, R.D. Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman and Hill, New York, 1992. [4] D.R. O’Reilly, L.K. Miller, V.A. Luckow, Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press, New York, 1992. [5] M.S. Kwon, T. Dojima, E.Y. Park, Comparative characterization of growth and recombinant protein production among three insect cell lines with four kinds of serum free media, Biotechnol. Bioprocess. Eng. 8 (2003) 142–146.
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N.G. Dalal et al. / Biochemical Engineering Journal 24 (2005) 27–30
[6] G.D. Pennock, C. Shoemaker, L.K. Miller, Strong and regulated expression of Escherichia coli -galactosidase in insect cells with a baculovirus vector, Mol. Cell Biol. 4 (1984) 399–406. [7] L.K. Miller, Baculoviruses: high-level expression in insect cells, Curr. Opin. Genet. Dev. 3 (1993) 97–101. [8] F. van der Wilk, J.W.M. van Lent, J.M. Vlak, Immunogold detection of polyhedrin, p10 and virion antigens in Autographa-californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells, J. Gen. Virol. 68 (1987) 2615–2623. [9] T.J. Wickham, T. Davis, R.R. Granados, M.L. Shuler, H.A. Wood, Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system, Biotechnol. Prog. 8 (1992) 391–396. [10] A.M. Crawford, L.K. Miller, Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus, J. Virol. 62 (1988) 2773–2781. [11] C.D. Richardson, M. Banville, M. Lalumiere, J. Vialard, E.A. Meighen, Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae, Intervirology 34 (1992) 213–227. [12] C.C. Jansson, M. Karp, C. Oker-Blom, J. Nasman, J.M. Savola, K.E. Akerman, Two human alpha 2-adrenoceptor subtypes alpha 2AC10 and alpha 2B-C2 expressed in Sf-9 cells couple to transduction pathway resulting in opposite effects on cAMP production, Eur. J. Pharmacol. 290 (1995) 75–83. [13] D.J. Sussman, 24-hour assay for estimating the titer of galactosidase-expressing baculovirus, BioTechniques 18 (1995) 50–51. [14] J.A. Mena, O.T. Ramirez, L.A. Palomares, Titration of non-occluded baculovirus using a cell viability assay, BioTechniques 34 (2003) 260–262.
[15] H.J. Cha, T. Gotoh, W.E. Bentley, Simplification of titer determination for recombinant baculovirus by green fluorescent protein marker, BioTechniques 23 (1997) 782–786. [16] H.J. Cha, N.G. Dalal, V.N. Vakharia, W.E. Bentley, Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells, J. Biotechnol. 69 (1999) 9–17. [17] M.D. Summers, G.E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedure. Texas agricultural experiment station bulletin no. 1555, 1987. [18] H.J. Cha, N.G. Dalal, M.Q. Pham, W.E. Bentley, Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand, Biotechnol. Prog. 15 (1999) 283–286. [19] H.J. Cha, N.G. Dalal, M.Q. Pham, V.N. Vakharia, G. Rao, W.E. Bentley, Insect larval expression process is optimized by generating fusions with green fluorescent protein, Biotechnol. Bioeng. 65 (1999) 316–324. [20] H.J. Cha, M.Q. Pham, G. Rao, W.E. Bentley, Expression of green fluorescent protein in insect larvae and its application for foreign protein production, Biotechnol. Bioeng. 56 (1997) 239–247. [21] P.D. Friesen, L.K. Miller, The regulation of baculovirus gene expression, Curr. Top Microbiol. Immunol. 131 (1986) 31–49. [22] F. Yang, L.G. Moss, G.N. Phillips Jr., The molecular structure of green fluorescent protein, Nature Biotechnol. 14 (1996) 1246–1251. [23] C.F. Chiang, D.T. Okou, T.B. Griffin, C.R. Verret, M.N.V. Williams, Green fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions, Arch. Biochem. Biophys. 394 (2001) 229–235. [24] M. Zimmer, Green fluorescent protein (GFP): application, structure, and related photophysical behavior, Chem. Rev. 102 (2002) 759–781.
Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter Nimish G. Dalal a , William E. Bentley a , Hyung Joon Cha b, ∗ b
a Center for Biosystems Research and Department of Chemical Engineering, University of Maryland, College Park, MD 20742, USA Department of Chemical Engineering and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea
Received 3 September 2004; received in revised form 17 January 2005; accepted 18 January 2005
Abstract A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) was constructed so that the green fluorescent protein (GFP) was produced via the early-to-late (ETL) promoter. This enabled rapid monitoring of the infection of Sf-9 insect cells. Notably, GFP and its fluorescence appeared ∼18 h prior to proteins expressed using very late polyhedrin (Polh) promoter. It is anticipated that the use of GFP under the control of ETL promoter will facilitate vector construction, virus isolation, and titer determination. © 2005 Elsevier B.V. All rights reserved. Keywords: Baculovirus infection; Early ETL promoter; Green fluorescent protein; Recombinant protein production
1. Introduction The baculovirus expression vector (BEV) system is both effective and convenient for the overproduction of recombinant proteins in eukaryotic cells [1–5]. The strong polyhedrin (Polh) promoter [6,7] and the 10 kDa fibrous polypeptide (P10) promoter [3,8], are both active in the very late phase of virus infection and when used to drive heterologous protein production can result in the accumulation of over 50% of the total protein [9]. The use of these promoters to establish the progress of infection and protein production would require waiting as long as 48 h post infection. Crawford and Miller [10] characterized the role of several early baculovirus genes on the expression of late viral genes. They also noted the -galactosidase produced under the early-to-late (ETL) promoter could be useful in aiding the identification of occlusionbody negative recombinants. Visualization of the infection process can be particularly difficult. The availability of a reporter protein can greatly facilitate developmental work ∗
Corresponding author. Tel.: +82 54 2792280; fax: +82 54 2795528. E-mail address: [email protected] (H.J. Cha).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.01.026
[11–13]. Richardson et al. developed a method, which uses the -galactosidase under control of early and late promoters including the ETL promoter to help aid in plaque assays [11]. Alternative techniques for virus titration continue to emerge [14]. In the previous work, we employed green fluorescent protein (GFP) as a monitoring reporter for a simple and rapid visualization of baculovirus, AcMNPV, infection [15,16]. In the present work, we have coupled, for the first time, the advantages of ETL promoter with GFP for a more simple and early monitoring of baculovirus infection.
2. Materials and methods 2.1. Construction of expression vector Several baculoviruses were constructed for this work. First, a pBlueBacHis2(lacZ) transfer vector was constructed by polymerase chain reaction (PCR) amplification around the lacZ gene sequence of the pBlueBacHis2/CAT transfer vector (Invitrogen, USA). The gfpuv gene was ex-
28
N.G. Dalal et al. / Biochemical Engineering Journal 24 (2005) 27–30
Fig. 1. Gene map of recombinant transfer vector pBBH(lacZ)GFPuv and pBBH-GFPuv/CAT. Abbreviation: PPH , polyhedrin promoter; PETL , early-to-late promoter; ColE1, Escherichia coli replication origin; Amp, ampicillin resistant gene; RS, recombination site; GFPuv, UV-optimized gfp gene; CAT, cat gene; (His)6 , histidine affinity ligand; EK, enterokinase cleavage site; lacZ, -galactosidase gene.
cised from the pGFPuv plasmid (Clontech, USA) using EagI and XbaI (Promega, USA) digestion and the gfpuv gene was inserted in frame under the ETL expression promoter resulting in a transfer vector denoted pBBH(lacZ)GFPuv (Fig. 1A). Then, a recombinant baculovirus, vPETL -GFPuv, was obtained by co-transfection of pBBH(lacZ)GFPuv with wild type AcNPV DNA (Bac-N-BlueTM baculovirus DNA, Invitrogen) into Sf-9 insect cells [17]. Successful cotransfection was observed through green fluorescence using a fluorescence microscope and virus amplification and titer were performed as described previously [15]. A pBBHGFPuv/CAT fusion transfer vector (Fig. 1B) was constructed from pBlueBacHis2-CAT (Invitrogen), containing the Polh promoter for expression of fusion foreign protein, GFP and chloramphenicol acetyl-transferase (CAT) [18,19]. Recombinant virus vPH-GFPuv/CAT was obtained and titered in a similar manner as vPETL -GFPuv. 2.2. Cell culture and sample preparation Sf-9 insect cells (ATCC, CRL-1711) were grown in SF900-II SFM (Invitrogen) and subcultured routinely every 3–4 days [4]. Experiments were performed on 230 ml cell cultures (250 ml spinner flasks) divided from single inocula grown at 27 ◦ C. Infection with the baculoviruses was performed during exponential growth (∼1 × 106 cells ml−1 ) with a predetermined volume of virus stock solution to yield a multiplicity of infection (MOI) of 5, at time denoted t = 0. Culture samples were collected initially every 6 h and then every 12 h after 60 h post infection (hpi). 2.3. Analytical methods Total cell counts were performed using a double chamber hemacytometer (Fisher Scientific, USA), and viability was determined by a Trypan blue (Sigma, USA) exclusion assay using a 0.4% solution. GFP was quantified using whole cell fraction without medium by fluorescence spectrometry
(MPF-66; Perkin-Elmer, USA) at an excitation wavelength of 395 nm and an emission wavelength of 509 nm. Also, GFP from whole cell fraction was assayed using Western blots. 2.4. Western blot analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by mixing a sample with sample buffer (0.5 M Tris–HCl, pH 6.8, 10% glycerol, 5% SDS, 5% -mercaptoethanol, and 0.25% bromophenol blue), incubating at 100 ◦ C for 3 min, centrifuging for 1 min, and loading onto a 15% slab gel. After electrophoresis, the gel was transferred onto a nitrocellulose membrane (Bio-Rad, USA) with a Bio-Rad Mini-Trans Blot Cell in Bjerrum and Schafer-Nielsen transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol; pH 9.2) for 20 min at 10 V followed by 20 min at 20 V. The nitrocellulose membrane was probed with 1:2000 dilution of polyclonal anti-rGFP antibody (Clontech), and detected with 1:5000 dilution of goat anti-rabbit IgG conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, USA) and BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) color development reagent (Sigma).
3. Results and discussion Suspended cell cultures (230 ml) were infected and monitored over the course of infection. Effectiveness of baculovirus infection depends on cell culture conditions such as virus titer, initial cell number, initial cell viability, and medium composition. It was already shown that there is a correlation between baculovirus infection efficiency and GFP production [15,19]. Uninfected cultures doubled in population number, increasing to a maximum of 2.6 × 106 cells ml−1 (data not shown). Cell densities for the infected cultures remained relatively constant post infection, near 1.5 × 106 cells ml−1 (data not shown). The viabilities for
N.G. Dalal et al. / Biochemical Engineering Journal 24 (2005) 27–30
29
Fig. 2. Western blot analyses of GFP expression under ETL () and Polh () promoters, (A) developed Western blots and (B) quantified results that were normalized to the first non-zero intensities from scanned images (12 hpi, ETL signal).
the infected cultures declined rapidly after 60 hpi, while the uninfected culture declined gradually to approximately 70% viability by the end of the cultures (data not shown). A time course for GFP production was examined via Western blot (Fig. 2), indicating a clear shift of ∼18 h for ETLdriven expression in comparison to that expressed via the Polh promoter. The ETL-driven GFP reached a maximum at 50 hpi, after which the accumulation level declined steadily. The decrease was possibly due to proteolysis [20] and/or cell lysis following virus infection, although the latter is less likely given the previously observed persistence of GFP from the very late Polh vectors. Under the Polh promoter, GFP was detected at approximately 42 hpi with a maximum at 96 hpi followed by a sharp decrease. These results correspond well to the expected profile, where immediate-early and delayedearly genes are expressed between 0 and 10 hpi, the transition to the late phase occurring between 10 and 15 hpi, and the very late genes expressed after 18 hpi [21]. Fig. 3A depicts the GFP fluorescence intensities over time. Consistent with Western blots, the GFP fluorescence intensity under the Polh promoter increased rapidly 42 hpi until reaching a maximum at ∼96 hpi, but without a marked decline. As the Western blots indicated a decrease from 96 to 108 h while fluorescence increased, we expect the loss of GFP on Western blot was most likely due to partial degradation without loss of the chromophore, as opposed to lysis, which would additionally diminish fluorescence. This is consistent with our previous study [16]. GFP has chromophore structure that is responsible for its fluorescence and protected by outside barrel (or -can) structure [22]. It was known that GFP could reveal fluorescence unless chromophore structure is not ruined even though full length of protein will be reduced due to -barrel structure breakage by proteolysis [23,24]. Under the early ETL promoter, GFP fluorescence remained low and fairly constant after 48 hpi, but increased initially near 18 hpi
Fig. 3. GFP fluorescence intensity for control (䊉) and cultures infected with vPETL -GFPuv () and vPH-GFPuv/CAT (), (A) fluorescence intensity over entire culture time and (B) fluorescence intensity for first 48 hpi.
(see insert Fig. 3B) resulting in a similar 18 h time shift observed by Western blot analysis.
4. Conclusions GFP production under the ETL promoter showed a clear shift of ∼18 h in comparison to that under the very late Polh promoter. Importantly, these results show that ∼18 h earlier expression of GFP under ETL promoter can facilitate more rapid monitoring in progress of recombinant baculovirus infection for application to virus construction, virus isolation, and titer determination.
References [1] L.K. Miller, Baculoviruses as gene expression vectors, Annu. Rev. Microbiol. 42 (1988) 177–199. [2] V.A. Luckow, M.D. Summers, Trends in the development of baculovirus expression vectors, Biotechnology 6 (1988) 47–55. [3] L.A. King, R.D. Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman and Hill, New York, 1992. [4] D.R. O’Reilly, L.K. Miller, V.A. Luckow, Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press, New York, 1992. [5] M.S. Kwon, T. Dojima, E.Y. Park, Comparative characterization of growth and recombinant protein production among three insect cell lines with four kinds of serum free media, Biotechnol. Bioprocess. Eng. 8 (2003) 142–146.
30
N.G. Dalal et al. / Biochemical Engineering Journal 24 (2005) 27–30
[6] G.D. Pennock, C. Shoemaker, L.K. Miller, Strong and regulated expression of Escherichia coli -galactosidase in insect cells with a baculovirus vector, Mol. Cell Biol. 4 (1984) 399–406. [7] L.K. Miller, Baculoviruses: high-level expression in insect cells, Curr. Opin. Genet. Dev. 3 (1993) 97–101. [8] F. van der Wilk, J.W.M. van Lent, J.M. Vlak, Immunogold detection of polyhedrin, p10 and virion antigens in Autographa-californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells, J. Gen. Virol. 68 (1987) 2615–2623. [9] T.J. Wickham, T. Davis, R.R. Granados, M.L. Shuler, H.A. Wood, Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system, Biotechnol. Prog. 8 (1992) 391–396. [10] A.M. Crawford, L.K. Miller, Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus, J. Virol. 62 (1988) 2773–2781. [11] C.D. Richardson, M. Banville, M. Lalumiere, J. Vialard, E.A. Meighen, Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae, Intervirology 34 (1992) 213–227. [12] C.C. Jansson, M. Karp, C. Oker-Blom, J. Nasman, J.M. Savola, K.E. Akerman, Two human alpha 2-adrenoceptor subtypes alpha 2AC10 and alpha 2B-C2 expressed in Sf-9 cells couple to transduction pathway resulting in opposite effects on cAMP production, Eur. J. Pharmacol. 290 (1995) 75–83. [13] D.J. Sussman, 24-hour assay for estimating the titer of galactosidase-expressing baculovirus, BioTechniques 18 (1995) 50–51. [14] J.A. Mena, O.T. Ramirez, L.A. Palomares, Titration of non-occluded baculovirus using a cell viability assay, BioTechniques 34 (2003) 260–262.
[15] H.J. Cha, T. Gotoh, W.E. Bentley, Simplification of titer determination for recombinant baculovirus by green fluorescent protein marker, BioTechniques 23 (1997) 782–786. [16] H.J. Cha, N.G. Dalal, V.N. Vakharia, W.E. Bentley, Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells, J. Biotechnol. 69 (1999) 9–17. [17] M.D. Summers, G.E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedure. Texas agricultural experiment station bulletin no. 1555, 1987. [18] H.J. Cha, N.G. Dalal, M.Q. Pham, W.E. Bentley, Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand, Biotechnol. Prog. 15 (1999) 283–286. [19] H.J. Cha, N.G. Dalal, M.Q. Pham, V.N. Vakharia, G. Rao, W.E. Bentley, Insect larval expression process is optimized by generating fusions with green fluorescent protein, Biotechnol. Bioeng. 65 (1999) 316–324. [20] H.J. Cha, M.Q. Pham, G. Rao, W.E. Bentley, Expression of green fluorescent protein in insect larvae and its application for foreign protein production, Biotechnol. Bioeng. 56 (1997) 239–247. [21] P.D. Friesen, L.K. Miller, The regulation of baculovirus gene expression, Curr. Top Microbiol. Immunol. 131 (1986) 31–49. [22] F. Yang, L.G. Moss, G.N. Phillips Jr., The molecular structure of green fluorescent protein, Nature Biotechnol. 14 (1996) 1246–1251. [23] C.F. Chiang, D.T. Okou, T.B. Griffin, C.R. Verret, M.N.V. Williams, Green fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions, Arch. Biochem. Biophys. 394 (2001) 229–235. [24] M. Zimmer, Green fluorescent protein (GFP): application, structure, and related photophysical behavior, Chem. Rev. 102 (2002) 759–781.