- Email: [email protected]
Rapid non-invasive monitoring of baculovirus infection for insect larvae using green fluorescent protein reporter under early-to-late promoter and a GFP-specific optical probe
Process Biochemistry 41 (2006) 947–950 www.elsevier.com/locate/procbio
Short communication
Rapid non-invasive monitoring of baculovirus infection for insect larvae using green fluorescent protein reporter under early-to-late promoter and a GFP-specific optical probe Nimish G. Dalal a, Hyung Joon Cha b,*, Shannon F. Kramer a, Yordan Kostov c, Govind Rao c, William E. Bentley a,** a
Center for Biosystems Research, Department of Chemical Engineering, University of Maryland, College Park, MD 20742, USA b Division of Molecular and Life Sciences, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c Medical Biotechnology Center, Department of Chemical and Biochemical Engineering, University of Maryland-Baltimore County, MD 21250, USA Received 6 April 2005; received in revised form 10 October 2005; accepted 15 October 2005
Abstract We investigated monitoring for baculovirus infection of insect Trichoplusia ni larvae by combining the green fluorescent protein (GFP) reporter, the early-to-late (ETL) baculoviral promoter, and a GFP-specific optical probe. Because GFP is under the ETL promoter control, it facilitated rapid monitoring of recombinant baculovirus infection on insect larva system. Employment of GFP-specific optical probe also enabled non-invasive and accurate GFP monitoring with 10 h prior to invasive and GFP Western blot assay. This combination may assist in monitoring protein production runs, determining optimal infection and harvest timing, and in general, help to increase the yield of recombinant proteins expressed in larvae. # 2005 Elsevier Ltd. All rights reserved. Keywords: Baculovirus infection; Insect larvae; Early-to-late promoter; Green fluorescent protein; GFP-specific optical probe; Non-invasive monitoring
1. Introduction Advances in recombinant DNA technology have made it possible to produce recombinant proteins that may not have been unachievable or unfeasible. Further, a variety of host and expression systems can be employed to produce the desired recombinant protein. To facilitate the expression of recombinant proteins in eukaryotes, virus systems have been gaining much attention. The baculovirus, Autographa califonica nuclear polyhedrosis virus (AcNPV), in particular, has been of important academic and industrial interest because of the advantages the system offers. These include correct functionality of the foreign protein [1,2], high expression levels [1,3], the possibility of post-translational modifications [4,5],
* Corresponding author. Tel.: +82 54 279 2280; fax: +82 54 279 5528. ** Corresponding author. Fax: +1 301 314 9075. E-mail addresses: [email protected] (H.J. Cha), [email protected] (W.E. Bentley). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.10.020
capacity for large DNA inserts, capacity for the expression of unspliced genes, simplicity, and simultaneous expressions of multiple genes. In general, the recombinant baculovirus is added to the insect cell culture during mid to late exponential phase, and the recombinant protein is expressed in high quantities, sometimes as great as 50% of the total cellular protein [6]. Because of the cost and bioreactor complexities, we have been studying the insect larvae/baculovirus expression system as a cost-effective alternative to the insect cell/baculovirus expression system [7–11]. Larvae have been used successfully for the production of a variety of foreign proteins at high levels [9,12–15]. Currently, the system entails synchronous infection with recombinant virus, either by feeding per os (pre-absorbed in diet) or by injection into the cuticle [16]. As the larvae grow, the virus replicates and eventually, the larvae melanize (a polyphenol oxidase-mediated process that results in a discoloration (browning) of the cuticle), and die. This results in the loss of recombinant protein [8]. The synchronous infection and synchronous harvesting is a key to obtain the maximum recombinant protein.
948
N.G. Dalal et al. / Process Biochemistry 41 (2006) 947–950
Our previous works have demonstrated the use of a fusion protein construct that contains the green fluorescent protein (GFP) fused to the recombinant protein of interest could be used as an effective indicator of product level [8,9,11] and facilitate purification [10] in insect larvae expression system. Larvae that produced GFP were easily distinguished from uninfected individuals by simply looking for fluorescence under UV light [8,9]. Further, the quantity of GFP directly related to the amount of recombinant protein [8,9,11] and facilitated the selection of elution fractions, which contain the recombinant product [10]. We also showed that a GFP-specific optical probe [17] can be successfully employed to quantitatively detect GFP level in insect larvae system [11]. By using a baculovirus early-to-late (ETL) promoter that is activated early in the infection cycle and is of medium strength [18] instead of a conventional very late polyhedrin (Polh) promoter that is of strong strength, we demonstrated that early monitoring of baculovirus infection for insect Sf-9 cell system is possible [19]. In the present work, we investigated non-invasive and rapid monitoring of baculovirus infection for insect Trichoplusia ni larvae system using the GFP-specific optical probe and the GFP reporter under the ETL promoter.
2. Materials and methods 2.1. Construction of recombinant baculovirus Recombinant baculovirus, nPETL–GFPuv, was obtained by co-transfection of pBBH(DlacZ)GFPuv transfer vector [19] (Fig. 1) with wild type AcNPV DNA (Bac-N-BlueTM baculovirus DNA, Invitrogen, USA) into Sf-9 insect cells. Successful co-transfection was observed through green fluorescence using a fluorescence microscope (Olympus, Japan). Recombinant virus stocks were propagated in Sf-9 cells in tissue culture flasks at 27 8C. Recombinant virus titer expressed as pfu (plaque forming units)/ml was obtained by the end-point dilution assay and calculated using the standard method (50% tissue-culture infectious dose; TCID50) with the relationship pfu = TCID50 0.69 [20].
Fig. 1. Gene map of recombinant transfer vector pBBH(DlacZ)GFPuv. 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.
2.2. Larval system and infection The cabbage looper, T. ni, insect larvae were hatched from eggs (Entopath, USA) in Styrofoam cups containing solid food (Entopath) at 30 8C. The larvae were monitored during the hatching process and each hour all the larvae that had hatched were placed in food cups and labeled as to the time of hatching. Fourth instar larvae were used for infection experiments. At 120–124 h after hatching, the larvae were infected with 5 ml of virus diluted so as to deliver 2 104 pfu per larvae. A 50 ml Hamilton syringe (Hamilton, USA) with a 27-gauge needle (Hamilton) was used for injection. The needle was inserted into the larvae at the front of the fourth set of prolegs just under the skin (0.1–0.25 cm deep).
2.3. Sample preparation and storage Three larvae were collected every 6 h post-infection (hpi) until the control pupated or the larvae became too liquefied to manipulate. Three identical experimental set-ups were performed in tandem. For the 0 hpi collection, six larvae were harvested from the control cups because no recombinant protein is produced immediately upon infection. Six larvae were also collected for the 6 and 12 hpi time points as larvae size is too small to yield enough homogenized sample for assays. Starting at 18 hpi, three larvae were harvested for each time point. Collected larvae were homogenized in 2.5 volumes of homogenization buffer (phosphate buffered saline (PBS) pH 7.0 containing, 6 mM dithiothreitol (DTT) and 0.5% Triton X-100), based on the assumption that the density is that of water, so that a 100 mg larva would be homogenized in 250 ml of buffer. The larvae were homogenized using a Tissue TearorTM (Biospec Products Inc., USA). Homogenates were divided into two fractions; a 500 ml homogenate sample was centrifuged at 12,000 g for 10 min at 4 8C. The supernatant was collected and aliquoted into 50 ml for protein assay and homogenized fluorescence readings, 100 ml for Western blot analysis, and 50 ml for further analysis if necessary. The aliquots and left over pellets and supernatants were stored at 80 8C until needed.
2.4. Analytical assays Each larva at the time of collection was individually weighed and then measured using the GFP-specific optical probe [17] using the experimental setup (Fig. 2) [11]. A Spectro Max Gemini Plate Reader with SoftMax Pro 3.1 software (Molecular Devices, USA) was used for GFP measurements of homogenized larvae. A 96-well assay plate (Costar, USA) was used to mix and measure samples. For each sample, 5 ml of homogenized larvae was added
Fig. 2. GFP-specific optical probe. The probe (a) was constructed on the basis of the GFP sensor described in Kostov et al. [17]. The signal is registered as volts using a digital multimeter (b). The probe was mounted onto a laboratory microscope with a movable stage (c) enabling incremental adjustment of the gap between the larva (d) and sensor [11].
N.G. Dalal et al. / Process Biochemistry 41 (2006) 947–950
949
to 195 ml of water and mixed well. The samples were done in triplicate, and the average of the three samples was used as the homogenized fluorescence. The total protein assay was performed using a protein assay kit (Bio-Rad Laboratory, USA) using bovine serum albumin as a standard. The quantity of GFP was determined using polyclonal anti-GFP antibody (Clontech, USA) and goat antirabbit IgG conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, USA) for Western blots. As a standard, 66 hpi larvae from infection and the sample were diluted 10-fold.
3. Results and discussion Injection-mediated infected larvae emitted green fluorescence when excited by UV light in contrast to the non-infected larvae (data not shown). This green fluorescence indicates that infection via the recombinant baculovirus has taken place and viral genes, as well as any transgenes carried by the recombinant virus are being expressed. Time courses of infected and non-infected larvae mass are depicted (Fig. 3A). The average larva mass increased continuously throughout the culture. The infected larva showed slowing in growth after 30 hpi and leveling off after 50 hpi. This appears to indicate that infection has commenced after about 30 hpi. Western blot analysis indicated that GFP under the ETL promoter was expressed at approximately 36 hpi (Fig. 3B). Band intensity data that were obtained by densitometry analysis showed quantifiable results after 42 hpi (Fig. 3B). Detection of GFP was also performed using a GFP-specific optical probe (Fig. 2). Using the optical probe, GFP was detected after 26 hpi (Fig. 3C) and the fluorescence readings increased until it leveled off after 50 hpi. This time profile of GFP agreed with homogenized larvae fluorescence intensities using a fluorescence spectrometer (data not shown) as already shown in the previous report having very late Polh promoter for GFP expression [11]. Importantly, we found that employment of non-invasive GFP-specific optical probe enabled rapid GFP monitoring with 10 h prior to invasive GFP Western blot that is relatively non-sensitive assay due to intrinsic experimental inefficiency [11]. In the previous report, it was shown that the error bars associated with the optical readings were 3–5-fold smaller than those of Western blots [11]. Even though different accuracies, these two methods showed quantitative correlation [11]. In our previous works, GFP as a fusion partner under a strong very late baculovirus promoter such as Polh did not show detectable levels of fluorescence until after 60 hpi when larvae were infected per os [9], or 36 hpi when larvae were infected via subcutaneous infection, as in the present study [11]. However, by employment of ETL promoter, we achieved approximately 10 h earlier monitoring of GFP expression. Even though lower expression level of GFP was obtained by medium strength ETL promoter, this was still sufficient to detect using the optical probe. Therefore, there is no apparent need to express the reporter at higher levels (which might independently affect product protein expression in non-fusion systems). This ETL promoter strategy for baculovirus infection was also successfully applied in suspended insect Sf-9 cell system [19]. In summary, expression of GFP reporter under the ETL promoter in T. ni larva was successfully detected using the
Fig. 3. Time profiles of (A) average larva mass, (B) normalized GFP band intensity from Western blot analysis, and (C) GFP from GFP-specific optical probe. Symbols: *, non-infected control; *, culture infected with nPETL– GFPuv. Each value and error bar represents the mean of three independent experiments (three larvae were collected for each value per each experiment. Thus, total nine larvae were assayed for each value) and its standard deviation.
previously described GFP-specific optical probe. Its expression using the ETL promoter was 10 h earlier than the previous case using the very late Polh promoter and its monitoring using non-invasive and accurate GFP-specific optical probe was possible 10 h prior to invasive Western blot assay. Because of these attributes, ETL-mediated GFP co-expression may facilitate more rapid monitoring of recombinant baculovirus infection of insect larvae as mini-bioreactors for foreign protein production, which in turn, might improve the yields of product proteins.
950
N.G. Dalal et al. / Process Biochemistry 41 (2006) 947–950
References [1] Luckow VA, Summers MD. Signals important for high-level expression of foreign genes in Autographa colifonica nuclear polyhedrosis virus expression vectors. Virology 1988;167:56–71. [2] Miller LK. Baculoviruses as gene expression vectors. Annu Rev Microbiol 1988;42:177–99. [3] Segura MM, Levin G, Miranda MV, Mendive FM, Targovnik HM, Cascone O. High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture. Process Biochem 2005;40:795–800. [4] Cameron IR, Possee RD, Bishop DHL. Insect cell-culture technology in baculovirus expression systems. Trends Biotechnol 1989;7:66–70. [5] Fraser MJ. Expression of eukaryotic genes in insect cultures. In Vitro Cell Dev Biol 1989;25:225–35. [6] Wickham TJ, Davis T, Granados RR, Shuler ML, Wood HA. Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol Prog 1992;8:391–6. [7] Pham MQ, Naggie S, Wier M, Cha HJ, Bentley WE. Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolysis. Biotechnol Bioeng 1999;62:175–82. [8] Cha HJ, Pham MQ, Rao G, Bentley WE. Expression of green fluorescent protein in insect larvae and its application for foreign protein production. Biotechnol Bioeng 1997;56:239–47. [9] Cha HJ, Dalal NG, Pham MQ, Vakharia VN, Rao G, Bentley WE. Insect larval expression process is optimized by generating fusions with green fluorescent protein. Biotechnol Bioeng 1999;65:316–24. [10] Cha HJ, Dalal NG, Pham MQ, Bentley WE. 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 1999;15:283–6.
[11] Kramer SF, Kostov Y, Rao G, Bentley WE. Ex vivo monitoring of protein production in baculovirus-infected Trichoplusia ni larvae with a GFPspecific optical probe. Biotechnol Bioeng 2003;83:241–7. [12] Maeda S, Kawai T, Obinata M, Fujiwara H, Horiuchi T, Saeki Y, Sato Y, Furusawa M. Production of human a-interferon in silkworm using a baculovirus vector. Nature 1985;315:592–4. [13] Kuroda K, Groner A, Frese K, Drenckhahn D, Hauser C, Rott R, Doerfler W, Klenk HD. Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J Virol 1989;63:1677–85. [14] Tremblay NM, Kennedy BP, Street IP, Kaupp WJ, Laliberte F, Weech PK. Human group ii phospholipase a2 expressed in Trichoplusia ni larvae— isolation and kinetic properties of the enzyme. Protein Expr Purif 1993; 4:490–8. [15] Lai SY, Lee MS, Chen HC, Shen PC, Jinn TR, Kao SS, Wang MY. Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein, rVP2H, in insect larvae. Process Biochem 2004;39:571–7. [16] King LA, Possee RD. The baculovirus expression system: a laboratory guide. Chapman & Hall; 1992. [17] Kostov Y, Albano CR, Rao G. All solid-state GFP sensor. Biotechnol Bioeng 2000;70:473–7. [18] Richardson CD, Banville M, Lalumiere M, Vialard J, Meighen EA. Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. Intervirology 1992;34:213–27. [19] Dalal NG, Bentley WE, Cha HJ. Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter. Biochem Eng J 2005;24:27–30. [20] O’Reilly DR, Miller LK, Luckow VA. Baculovirus expression vectors: a laboratory manual. Oxford University Press; 1994.
Short communication
Rapid non-invasive monitoring of baculovirus infection for insect larvae using green fluorescent protein reporter under early-to-late promoter and a GFP-specific optical probe Nimish G. Dalal a, Hyung Joon Cha b,*, Shannon F. Kramer a, Yordan Kostov c, Govind Rao c, William E. Bentley a,** a
Center for Biosystems Research, Department of Chemical Engineering, University of Maryland, College Park, MD 20742, USA b Division of Molecular and Life Sciences, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c Medical Biotechnology Center, Department of Chemical and Biochemical Engineering, University of Maryland-Baltimore County, MD 21250, USA Received 6 April 2005; received in revised form 10 October 2005; accepted 15 October 2005
Abstract We investigated monitoring for baculovirus infection of insect Trichoplusia ni larvae by combining the green fluorescent protein (GFP) reporter, the early-to-late (ETL) baculoviral promoter, and a GFP-specific optical probe. Because GFP is under the ETL promoter control, it facilitated rapid monitoring of recombinant baculovirus infection on insect larva system. Employment of GFP-specific optical probe also enabled non-invasive and accurate GFP monitoring with 10 h prior to invasive and GFP Western blot assay. This combination may assist in monitoring protein production runs, determining optimal infection and harvest timing, and in general, help to increase the yield of recombinant proteins expressed in larvae. # 2005 Elsevier Ltd. All rights reserved. Keywords: Baculovirus infection; Insect larvae; Early-to-late promoter; Green fluorescent protein; GFP-specific optical probe; Non-invasive monitoring
1. Introduction Advances in recombinant DNA technology have made it possible to produce recombinant proteins that may not have been unachievable or unfeasible. Further, a variety of host and expression systems can be employed to produce the desired recombinant protein. To facilitate the expression of recombinant proteins in eukaryotes, virus systems have been gaining much attention. The baculovirus, Autographa califonica nuclear polyhedrosis virus (AcNPV), in particular, has been of important academic and industrial interest because of the advantages the system offers. These include correct functionality of the foreign protein [1,2], high expression levels [1,3], the possibility of post-translational modifications [4,5],
* Corresponding author. Tel.: +82 54 279 2280; fax: +82 54 279 5528. ** Corresponding author. Fax: +1 301 314 9075. E-mail addresses: [email protected] (H.J. Cha), [email protected] (W.E. Bentley). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.10.020
capacity for large DNA inserts, capacity for the expression of unspliced genes, simplicity, and simultaneous expressions of multiple genes. In general, the recombinant baculovirus is added to the insect cell culture during mid to late exponential phase, and the recombinant protein is expressed in high quantities, sometimes as great as 50% of the total cellular protein [6]. Because of the cost and bioreactor complexities, we have been studying the insect larvae/baculovirus expression system as a cost-effective alternative to the insect cell/baculovirus expression system [7–11]. Larvae have been used successfully for the production of a variety of foreign proteins at high levels [9,12–15]. Currently, the system entails synchronous infection with recombinant virus, either by feeding per os (pre-absorbed in diet) or by injection into the cuticle [16]. As the larvae grow, the virus replicates and eventually, the larvae melanize (a polyphenol oxidase-mediated process that results in a discoloration (browning) of the cuticle), and die. This results in the loss of recombinant protein [8]. The synchronous infection and synchronous harvesting is a key to obtain the maximum recombinant protein.
948
N.G. Dalal et al. / Process Biochemistry 41 (2006) 947–950
Our previous works have demonstrated the use of a fusion protein construct that contains the green fluorescent protein (GFP) fused to the recombinant protein of interest could be used as an effective indicator of product level [8,9,11] and facilitate purification [10] in insect larvae expression system. Larvae that produced GFP were easily distinguished from uninfected individuals by simply looking for fluorescence under UV light [8,9]. Further, the quantity of GFP directly related to the amount of recombinant protein [8,9,11] and facilitated the selection of elution fractions, which contain the recombinant product [10]. We also showed that a GFP-specific optical probe [17] can be successfully employed to quantitatively detect GFP level in insect larvae system [11]. By using a baculovirus early-to-late (ETL) promoter that is activated early in the infection cycle and is of medium strength [18] instead of a conventional very late polyhedrin (Polh) promoter that is of strong strength, we demonstrated that early monitoring of baculovirus infection for insect Sf-9 cell system is possible [19]. In the present work, we investigated non-invasive and rapid monitoring of baculovirus infection for insect Trichoplusia ni larvae system using the GFP-specific optical probe and the GFP reporter under the ETL promoter.
2. Materials and methods 2.1. Construction of recombinant baculovirus Recombinant baculovirus, nPETL–GFPuv, was obtained by co-transfection of pBBH(DlacZ)GFPuv transfer vector [19] (Fig. 1) with wild type AcNPV DNA (Bac-N-BlueTM baculovirus DNA, Invitrogen, USA) into Sf-9 insect cells. Successful co-transfection was observed through green fluorescence using a fluorescence microscope (Olympus, Japan). Recombinant virus stocks were propagated in Sf-9 cells in tissue culture flasks at 27 8C. Recombinant virus titer expressed as pfu (plaque forming units)/ml was obtained by the end-point dilution assay and calculated using the standard method (50% tissue-culture infectious dose; TCID50) with the relationship pfu = TCID50 0.69 [20].
Fig. 1. Gene map of recombinant transfer vector pBBH(DlacZ)GFPuv. 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.
2.2. Larval system and infection The cabbage looper, T. ni, insect larvae were hatched from eggs (Entopath, USA) in Styrofoam cups containing solid food (Entopath) at 30 8C. The larvae were monitored during the hatching process and each hour all the larvae that had hatched were placed in food cups and labeled as to the time of hatching. Fourth instar larvae were used for infection experiments. At 120–124 h after hatching, the larvae were infected with 5 ml of virus diluted so as to deliver 2 104 pfu per larvae. A 50 ml Hamilton syringe (Hamilton, USA) with a 27-gauge needle (Hamilton) was used for injection. The needle was inserted into the larvae at the front of the fourth set of prolegs just under the skin (0.1–0.25 cm deep).
2.3. Sample preparation and storage Three larvae were collected every 6 h post-infection (hpi) until the control pupated or the larvae became too liquefied to manipulate. Three identical experimental set-ups were performed in tandem. For the 0 hpi collection, six larvae were harvested from the control cups because no recombinant protein is produced immediately upon infection. Six larvae were also collected for the 6 and 12 hpi time points as larvae size is too small to yield enough homogenized sample for assays. Starting at 18 hpi, three larvae were harvested for each time point. Collected larvae were homogenized in 2.5 volumes of homogenization buffer (phosphate buffered saline (PBS) pH 7.0 containing, 6 mM dithiothreitol (DTT) and 0.5% Triton X-100), based on the assumption that the density is that of water, so that a 100 mg larva would be homogenized in 250 ml of buffer. The larvae were homogenized using a Tissue TearorTM (Biospec Products Inc., USA). Homogenates were divided into two fractions; a 500 ml homogenate sample was centrifuged at 12,000 g for 10 min at 4 8C. The supernatant was collected and aliquoted into 50 ml for protein assay and homogenized fluorescence readings, 100 ml for Western blot analysis, and 50 ml for further analysis if necessary. The aliquots and left over pellets and supernatants were stored at 80 8C until needed.
2.4. Analytical assays Each larva at the time of collection was individually weighed and then measured using the GFP-specific optical probe [17] using the experimental setup (Fig. 2) [11]. A Spectro Max Gemini Plate Reader with SoftMax Pro 3.1 software (Molecular Devices, USA) was used for GFP measurements of homogenized larvae. A 96-well assay plate (Costar, USA) was used to mix and measure samples. For each sample, 5 ml of homogenized larvae was added
Fig. 2. GFP-specific optical probe. The probe (a) was constructed on the basis of the GFP sensor described in Kostov et al. [17]. The signal is registered as volts using a digital multimeter (b). The probe was mounted onto a laboratory microscope with a movable stage (c) enabling incremental adjustment of the gap between the larva (d) and sensor [11].
N.G. Dalal et al. / Process Biochemistry 41 (2006) 947–950
949
to 195 ml of water and mixed well. The samples were done in triplicate, and the average of the three samples was used as the homogenized fluorescence. The total protein assay was performed using a protein assay kit (Bio-Rad Laboratory, USA) using bovine serum albumin as a standard. The quantity of GFP was determined using polyclonal anti-GFP antibody (Clontech, USA) and goat antirabbit IgG conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, USA) for Western blots. As a standard, 66 hpi larvae from infection and the sample were diluted 10-fold.
3. Results and discussion Injection-mediated infected larvae emitted green fluorescence when excited by UV light in contrast to the non-infected larvae (data not shown). This green fluorescence indicates that infection via the recombinant baculovirus has taken place and viral genes, as well as any transgenes carried by the recombinant virus are being expressed. Time courses of infected and non-infected larvae mass are depicted (Fig. 3A). The average larva mass increased continuously throughout the culture. The infected larva showed slowing in growth after 30 hpi and leveling off after 50 hpi. This appears to indicate that infection has commenced after about 30 hpi. Western blot analysis indicated that GFP under the ETL promoter was expressed at approximately 36 hpi (Fig. 3B). Band intensity data that were obtained by densitometry analysis showed quantifiable results after 42 hpi (Fig. 3B). Detection of GFP was also performed using a GFP-specific optical probe (Fig. 2). Using the optical probe, GFP was detected after 26 hpi (Fig. 3C) and the fluorescence readings increased until it leveled off after 50 hpi. This time profile of GFP agreed with homogenized larvae fluorescence intensities using a fluorescence spectrometer (data not shown) as already shown in the previous report having very late Polh promoter for GFP expression [11]. Importantly, we found that employment of non-invasive GFP-specific optical probe enabled rapid GFP monitoring with 10 h prior to invasive GFP Western blot that is relatively non-sensitive assay due to intrinsic experimental inefficiency [11]. In the previous report, it was shown that the error bars associated with the optical readings were 3–5-fold smaller than those of Western blots [11]. Even though different accuracies, these two methods showed quantitative correlation [11]. In our previous works, GFP as a fusion partner under a strong very late baculovirus promoter such as Polh did not show detectable levels of fluorescence until after 60 hpi when larvae were infected per os [9], or 36 hpi when larvae were infected via subcutaneous infection, as in the present study [11]. However, by employment of ETL promoter, we achieved approximately 10 h earlier monitoring of GFP expression. Even though lower expression level of GFP was obtained by medium strength ETL promoter, this was still sufficient to detect using the optical probe. Therefore, there is no apparent need to express the reporter at higher levels (which might independently affect product protein expression in non-fusion systems). This ETL promoter strategy for baculovirus infection was also successfully applied in suspended insect Sf-9 cell system [19]. In summary, expression of GFP reporter under the ETL promoter in T. ni larva was successfully detected using the
Fig. 3. Time profiles of (A) average larva mass, (B) normalized GFP band intensity from Western blot analysis, and (C) GFP from GFP-specific optical probe. Symbols: *, non-infected control; *, culture infected with nPETL– GFPuv. Each value and error bar represents the mean of three independent experiments (three larvae were collected for each value per each experiment. Thus, total nine larvae were assayed for each value) and its standard deviation.
previously described GFP-specific optical probe. Its expression using the ETL promoter was 10 h earlier than the previous case using the very late Polh promoter and its monitoring using non-invasive and accurate GFP-specific optical probe was possible 10 h prior to invasive Western blot assay. Because of these attributes, ETL-mediated GFP co-expression may facilitate more rapid monitoring of recombinant baculovirus infection of insect larvae as mini-bioreactors for foreign protein production, which in turn, might improve the yields of product proteins.
950
N.G. Dalal et al. / Process Biochemistry 41 (2006) 947–950
References [1] Luckow VA, Summers MD. Signals important for high-level expression of foreign genes in Autographa colifonica nuclear polyhedrosis virus expression vectors. Virology 1988;167:56–71. [2] Miller LK. Baculoviruses as gene expression vectors. Annu Rev Microbiol 1988;42:177–99. [3] Segura MM, Levin G, Miranda MV, Mendive FM, Targovnik HM, Cascone O. High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture. Process Biochem 2005;40:795–800. [4] Cameron IR, Possee RD, Bishop DHL. Insect cell-culture technology in baculovirus expression systems. Trends Biotechnol 1989;7:66–70. [5] Fraser MJ. Expression of eukaryotic genes in insect cultures. In Vitro Cell Dev Biol 1989;25:225–35. [6] Wickham TJ, Davis T, Granados RR, Shuler ML, Wood HA. Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol Prog 1992;8:391–6. [7] Pham MQ, Naggie S, Wier M, Cha HJ, Bentley WE. Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolysis. Biotechnol Bioeng 1999;62:175–82. [8] Cha HJ, Pham MQ, Rao G, Bentley WE. Expression of green fluorescent protein in insect larvae and its application for foreign protein production. Biotechnol Bioeng 1997;56:239–47. [9] Cha HJ, Dalal NG, Pham MQ, Vakharia VN, Rao G, Bentley WE. Insect larval expression process is optimized by generating fusions with green fluorescent protein. Biotechnol Bioeng 1999;65:316–24. [10] Cha HJ, Dalal NG, Pham MQ, Bentley WE. 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 1999;15:283–6.
[11] Kramer SF, Kostov Y, Rao G, Bentley WE. Ex vivo monitoring of protein production in baculovirus-infected Trichoplusia ni larvae with a GFPspecific optical probe. Biotechnol Bioeng 2003;83:241–7. [12] Maeda S, Kawai T, Obinata M, Fujiwara H, Horiuchi T, Saeki Y, Sato Y, Furusawa M. Production of human a-interferon in silkworm using a baculovirus vector. Nature 1985;315:592–4. [13] Kuroda K, Groner A, Frese K, Drenckhahn D, Hauser C, Rott R, Doerfler W, Klenk HD. Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J Virol 1989;63:1677–85. [14] Tremblay NM, Kennedy BP, Street IP, Kaupp WJ, Laliberte F, Weech PK. Human group ii phospholipase a2 expressed in Trichoplusia ni larvae— isolation and kinetic properties of the enzyme. Protein Expr Purif 1993; 4:490–8. [15] Lai SY, Lee MS, Chen HC, Shen PC, Jinn TR, Kao SS, Wang MY. Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein, rVP2H, in insect larvae. Process Biochem 2004;39:571–7. [16] King LA, Possee RD. The baculovirus expression system: a laboratory guide. Chapman & Hall; 1992. [17] Kostov Y, Albano CR, Rao G. All solid-state GFP sensor. Biotechnol Bioeng 2000;70:473–7. [18] Richardson CD, Banville M, Lalumiere M, Vialard J, Meighen EA. Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. Intervirology 1992;34:213–27. [19] Dalal NG, Bentley WE, Cha HJ. Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter. Biochem Eng J 2005;24:27–30. [20] O’Reilly DR, Miller LK, Luckow VA. Baculovirus expression vectors: a laboratory manual. Oxford University Press; 1994.