CHAPTER FIFTEEN
Semliki Forest Virus-Based Expression of Recombinant GPCRs Kenneth Lundstrom1 PanTherapeutics, Lutry, Switzerland 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. SFV Expression System 2.1 Cell cultures 2.2 Viral vectors 2.3 Production of recombinant SFV particles 3. Expression of GPCRs 3.1 Drug discovery 3.2 Neuroscience 3.3 Structural biology 4. Summary References
332 333 334 334 335 339 339 342 346 346 348
Abstract Due to their importance as targets for drug development, rapid and consistent highlevel production of G protein-coupled receptors (GPCRs) has become an essential part of drug discovery. Alphaviruses, particularly recombinant Semliki Forest virus (SFV) particles, have provided the means for expression of a number of GPCRs in a broad range of mammalian host cell lines for pharmacological characterization by determination of receptor binding activity and functional coupling to G proteins. The rapid high-titer virus particle production has made it possible to study a large number of GPCRs in parallel. Moreover, large-scale production in adherent and suspension cultures of mammalian cells has provided sufficient amounts of GPCRs for purification and subsequent structural studies. Furthermore, the high preference for neuronal delivery of SFV particles has allowed functional and localization studies of recombinant proteins in hippocampal slice cultures, in primary neurons, and in vivo.
Methods in Enzymology, Volume 556 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.11.047
#
2015 Elsevier Inc. All rights reserved.
331
332
Kenneth Lundstrom
1. INTRODUCTION G protein-coupled receptors (GPCRs) represent the largest group of drug targets today and have therefore been subjected to extensive research (Lundstrom, 2006a). In this context, one key component has been robust and reproducible production of large amounts of high-quality recombinant GPCRs. In attempts to achieve this, a number of expression systems have been developed based on bacterial, yeast, insect, and mammalian cells (Lundstrom, 2006b). More recently, cell-free production of GPCRs has become a reality (Wang, Wang, & Ge, 2013). Generally, prokaryotic systems have been favored for recombinant protein expression due to their simple use and inexpensive large-scale production (Tucker & Grisshammer, 1996). However, the drawback of applying bacterial systems has been the lack of appropriate posttranslational modifications of the expressed recombinant proteins. Therefore, the attention has been shifted to yeast-based systems, especially to recombinant protein expression in Pichia pastoris (Weiss, Haase, Michel, & Reilander, 1998). Likewise, baculovirus-based expression of GPCRs has seen much success (Possee, 1997). Although GPCRs expressed in both yeast and insect cell systems present differences related to glycosylation patterns in mammalian cells, especially the latter has provided high yields of functional GPCRs. Particularly, encouraging has been the success in purification and structural characterization of several GPCRs expressed in insect cells (Cherezov et al., 2007; Granier et al., 2012; Jaakola & Ijzerman, 2010; Warne et al., 2008). Until recently, application of transiently (Girard et al., 2001) and stably (Lohse, 1992) transfected mammalian cells for GPCR expression has been problematic due to low receptor yields and time- and labor-consuming procedures. However, current technology improvement has allowed enhanced expression of recombinant proteins, including transmembrane receptors, at levels competitive to what has been achieved in insect cells (Hassaine et al., 2013). One alternative for GPCR expression in mammalian cells has been the application of alphavirus vectors (Lundstrom, 2003). Especially, replicationdeficient Semliki Forest virus (SFV) particles have provided high-level expression of a large number of GPCRs in a broad range of mammalian host cells (Hassaine et al., 2006). In this chapter, the use of the alphavirus expression system is described, and a range of applications in drug discovery and neuroscience are presented.
SFV-Based Expression of Recombinant GPCRs
333
2. SFV EXPRESSION SYSTEM Alphavirus expression vectors have been engineered for SFV (Liljestr€ om & Garoff, 1991), Sindbis virus (SIN) (Xiong et al., 1989), and Venezuelan equine encephalitis virus (Davis, Brown, & Johnston, 1989). Generally, three variations of vector systems exist (Fig. 1): (1) Replicationdeficient particles are generated from in vitro-transcribed RNA originating from the expression vector carrying four nonstructural (replicase) genes, the gene of interest, and a helper vector providing the viral structural genes (Liljestr€ om & Garoff, 1991). The generated RNA molecules are introduced by electroporation/transfection into baby hamster kidney (BHK) cells.
Figure 1 SFV-based expression systems. (A) Replication-deficient particles generated from in vitro-transcribed RNA from expression and helper vector DNA introduced into BHK cells by electroporation or lipofection methods. Recombinant particles are able to infect mammalian cells for recombinant GPCR expression. (B) Replication-proficient particles generated from in vitro-transcribed RNA from the full-length SFV genome and the gene of interest as above with the difference that generated particles produce new virus progeny. (C) DNA-layered vectors, where the SP6 RNA polymerase promoter has been replaced by a CMV promoter (▲), which allows transfection of mammalian cells with plasmid DNA for GPCR expression.
334
Kenneth Lundstrom
Due to the presence of a packaging signal in the replicase gene region, only RNA originating from the expression will be packaged into alphavirus particles. As these particles lack any structural genes, it will allow only one round of infection of host cells resulting in high levels of transgene expression. However, no further generation of viral progeny takes place. (2) Replication-proficient particles are produced from in vitro-transcribed RNA comprising the full-length alphavirus genome with an additional subgenomic promoter and the foreign gene of interest (Va¨ha¨-Koskela et al., 2003). Viral particles generated in BHK cells are capable of producing virus progeny upon infection of mammalian cells. Simultaneously, high-level transgene expression will occur. (3) DNA-layered vectors are based on the alphavirus expression vector where the SP6 or T7 promoter used for in vitro transcription has been replaced by a CMV promoter (Berglund, Smerdou, Fleeton, Tubulekas, & Liljestr€ om, 1998). In this case, the transgene expression relies on transfection methods for delivery to mammalian cells. On the contrary, no viral particles are produced at any stage of the procedure.
2.1 Cell cultures BHK-21 cells (ATCC CCL-10), commonly used as the host cell line for the production of alphavirus particles, are cultured in a 1:1 mixture of Dulbecco’s modified F-12 medium and Iscove’s modified Dulbecco’s medium supplemented with 4 mM glutamate and 10% fetal calf serum (FCS). Similarly, CHO-K1 (Chinese hamster ovary) and HEK293 (human embryonic kidney) cells applied as host cell lines for GPCR expression can be cultured in the same media, whereas C8166 (human T lymphocyte) cells are grown in RPI Medium, 4 mM glutamate, and 10% FCS.
2.2 Viral vectors The SFV vectors commonly used for recombinant particle generation comprise of pSFVgen (also pSFV1) expression vector and the second-generation pSFV-Helper2 (Berglund et al., 1993). For SIN, two alternative expression vectors, pSINrep5 and pSINrep504 with a capsid enhancer sequence (Frolov & Schlesinger, 1996), and SIN-DH-EB and SIN-DH-BB helper vectors (Bredenbeek, Frolov, Rice, & Schlesinger, 1993) are used. In this review, the focus is on SFV vectors although similar methods are applied for other alphavirus vectors.
SFV-Based Expression of Recombinant GPCRs
335
2.3 Production of recombinant SFV particles Replication-deficient particles, especially based on SFV vectors, have been frequently used for recombinant protein expression in a large number of mammalian cell lines (Lundstrom, 2003), in primary neurons (Lundstrom & Ehrengruber, 2003), in hippocampal slice cultures (Ehrengruber et al., 1999), and in vivo (Lundstrom, Richards, Pink, & Jenck, 1999). In principle, the basic method for generation of recombinant SFV particles is the same independent of further application as briefly described below. General cloning methods are applied for the subcloning of foreign genes into the SFV expression vector. After clone verification, DNA Midipreps or Maxipreps are prepared and linearized with SpeI, SapI (pSFV1 and pSFVHelper2) or NruI (pSFV2gen) prior to in vitro transcription. Generally, 5–10 μg plasmid DNA (larger quantities can be stored at -20 °C) is linearized, purified by phenol/chloroform extraction followed by ethanol precipitation. DNA pellets are resuspended in RNase-free H2O at a final concentration of 0.5 μg/μL. Alternatively, MicroSpin™ S-200 HR Columns (Amersham) can be applied according to the manufacturer’s instructions. 2.3.1 In vitro transcription High-titer virus production requires high-quality in vitro-transcribed RNA. Although manufacturers provide their own in vitro transcription kits including their SP6 buffer, it is recommended to use the optimized buffer below. To avoid precipitation due to the presence of spermidine, the in vitro transcription reactions should be set up at room temperature. Enzyme components should be added last. 5 μL (2.5 μg) linearized plasmid DNA 5 μL 10 SP6 buffer (400 mM HEPES, pH 7.4, 60 mM magnesium acetate, 20 mM spermidine) 5 μL 10 mM m7G(50 )ppp(50 )G 5 μL 50 mM DTT 5 μL rNTP mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, 5 mM rGTP) x μL RNase-free H2O to reach a final volume of 50 μL 2 μL (50 U/μL) RNase inhibitor 5 μL (20 U/μL) SP6 RNA polymerase After 1 h incubation at 37 °C 1–4 μL aliquots are loaded on a 0.8% agarose gel for RNA quality evaluation, where thick bands without smearing
336
Kenneth Lundstrom
indicate high-quality RNA. Generated RNA molecules can be directly used for electroporations or stored for weeks at -80 °C. 2.3.2 Electroporation of RNA BHK-21 cells, commonly used for production of high-titer SFV stocks, should only possess a low passage number (cultured less than 3 months) and no more than 80% confluency to provide high viability. Cells are washed once with PBS and trypsinized with 6 mL trypsin–EDTA per T175 flask for 5 min at 37 °C. Clumps are removed by resuspension and medium added to 25 mL. After centrifugation at 800 g for 5 min, the cell pellet is resuspended in a small volume (
Semliki Forest Virus-Based Expression of Recombinant GPCRs Kenneth Lundstrom1 PanTherapeutics, Lutry, Switzerland 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. SFV Expression System 2.1 Cell cultures 2.2 Viral vectors 2.3 Production of recombinant SFV particles 3. Expression of GPCRs 3.1 Drug discovery 3.2 Neuroscience 3.3 Structural biology 4. Summary References
332 333 334 334 335 339 339 342 346 346 348
Abstract Due to their importance as targets for drug development, rapid and consistent highlevel production of G protein-coupled receptors (GPCRs) has become an essential part of drug discovery. Alphaviruses, particularly recombinant Semliki Forest virus (SFV) particles, have provided the means for expression of a number of GPCRs in a broad range of mammalian host cell lines for pharmacological characterization by determination of receptor binding activity and functional coupling to G proteins. The rapid high-titer virus particle production has made it possible to study a large number of GPCRs in parallel. Moreover, large-scale production in adherent and suspension cultures of mammalian cells has provided sufficient amounts of GPCRs for purification and subsequent structural studies. Furthermore, the high preference for neuronal delivery of SFV particles has allowed functional and localization studies of recombinant proteins in hippocampal slice cultures, in primary neurons, and in vivo.
Methods in Enzymology, Volume 556 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.11.047
#
2015 Elsevier Inc. All rights reserved.
331
332
Kenneth Lundstrom
1. INTRODUCTION G protein-coupled receptors (GPCRs) represent the largest group of drug targets today and have therefore been subjected to extensive research (Lundstrom, 2006a). In this context, one key component has been robust and reproducible production of large amounts of high-quality recombinant GPCRs. In attempts to achieve this, a number of expression systems have been developed based on bacterial, yeast, insect, and mammalian cells (Lundstrom, 2006b). More recently, cell-free production of GPCRs has become a reality (Wang, Wang, & Ge, 2013). Generally, prokaryotic systems have been favored for recombinant protein expression due to their simple use and inexpensive large-scale production (Tucker & Grisshammer, 1996). However, the drawback of applying bacterial systems has been the lack of appropriate posttranslational modifications of the expressed recombinant proteins. Therefore, the attention has been shifted to yeast-based systems, especially to recombinant protein expression in Pichia pastoris (Weiss, Haase, Michel, & Reilander, 1998). Likewise, baculovirus-based expression of GPCRs has seen much success (Possee, 1997). Although GPCRs expressed in both yeast and insect cell systems present differences related to glycosylation patterns in mammalian cells, especially the latter has provided high yields of functional GPCRs. Particularly, encouraging has been the success in purification and structural characterization of several GPCRs expressed in insect cells (Cherezov et al., 2007; Granier et al., 2012; Jaakola & Ijzerman, 2010; Warne et al., 2008). Until recently, application of transiently (Girard et al., 2001) and stably (Lohse, 1992) transfected mammalian cells for GPCR expression has been problematic due to low receptor yields and time- and labor-consuming procedures. However, current technology improvement has allowed enhanced expression of recombinant proteins, including transmembrane receptors, at levels competitive to what has been achieved in insect cells (Hassaine et al., 2013). One alternative for GPCR expression in mammalian cells has been the application of alphavirus vectors (Lundstrom, 2003). Especially, replicationdeficient Semliki Forest virus (SFV) particles have provided high-level expression of a large number of GPCRs in a broad range of mammalian host cells (Hassaine et al., 2006). In this chapter, the use of the alphavirus expression system is described, and a range of applications in drug discovery and neuroscience are presented.
SFV-Based Expression of Recombinant GPCRs
333
2. SFV EXPRESSION SYSTEM Alphavirus expression vectors have been engineered for SFV (Liljestr€ om & Garoff, 1991), Sindbis virus (SIN) (Xiong et al., 1989), and Venezuelan equine encephalitis virus (Davis, Brown, & Johnston, 1989). Generally, three variations of vector systems exist (Fig. 1): (1) Replicationdeficient particles are generated from in vitro-transcribed RNA originating from the expression vector carrying four nonstructural (replicase) genes, the gene of interest, and a helper vector providing the viral structural genes (Liljestr€ om & Garoff, 1991). The generated RNA molecules are introduced by electroporation/transfection into baby hamster kidney (BHK) cells.
Figure 1 SFV-based expression systems. (A) Replication-deficient particles generated from in vitro-transcribed RNA from expression and helper vector DNA introduced into BHK cells by electroporation or lipofection methods. Recombinant particles are able to infect mammalian cells for recombinant GPCR expression. (B) Replication-proficient particles generated from in vitro-transcribed RNA from the full-length SFV genome and the gene of interest as above with the difference that generated particles produce new virus progeny. (C) DNA-layered vectors, where the SP6 RNA polymerase promoter has been replaced by a CMV promoter (▲), which allows transfection of mammalian cells with plasmid DNA for GPCR expression.
334
Kenneth Lundstrom
Due to the presence of a packaging signal in the replicase gene region, only RNA originating from the expression will be packaged into alphavirus particles. As these particles lack any structural genes, it will allow only one round of infection of host cells resulting in high levels of transgene expression. However, no further generation of viral progeny takes place. (2) Replication-proficient particles are produced from in vitro-transcribed RNA comprising the full-length alphavirus genome with an additional subgenomic promoter and the foreign gene of interest (Va¨ha¨-Koskela et al., 2003). Viral particles generated in BHK cells are capable of producing virus progeny upon infection of mammalian cells. Simultaneously, high-level transgene expression will occur. (3) DNA-layered vectors are based on the alphavirus expression vector where the SP6 or T7 promoter used for in vitro transcription has been replaced by a CMV promoter (Berglund, Smerdou, Fleeton, Tubulekas, & Liljestr€ om, 1998). In this case, the transgene expression relies on transfection methods for delivery to mammalian cells. On the contrary, no viral particles are produced at any stage of the procedure.
2.1 Cell cultures BHK-21 cells (ATCC CCL-10), commonly used as the host cell line for the production of alphavirus particles, are cultured in a 1:1 mixture of Dulbecco’s modified F-12 medium and Iscove’s modified Dulbecco’s medium supplemented with 4 mM glutamate and 10% fetal calf serum (FCS). Similarly, CHO-K1 (Chinese hamster ovary) and HEK293 (human embryonic kidney) cells applied as host cell lines for GPCR expression can be cultured in the same media, whereas C8166 (human T lymphocyte) cells are grown in RPI Medium, 4 mM glutamate, and 10% FCS.
2.2 Viral vectors The SFV vectors commonly used for recombinant particle generation comprise of pSFVgen (also pSFV1) expression vector and the second-generation pSFV-Helper2 (Berglund et al., 1993). For SIN, two alternative expression vectors, pSINrep5 and pSINrep504 with a capsid enhancer sequence (Frolov & Schlesinger, 1996), and SIN-DH-EB and SIN-DH-BB helper vectors (Bredenbeek, Frolov, Rice, & Schlesinger, 1993) are used. In this review, the focus is on SFV vectors although similar methods are applied for other alphavirus vectors.
SFV-Based Expression of Recombinant GPCRs
335
2.3 Production of recombinant SFV particles Replication-deficient particles, especially based on SFV vectors, have been frequently used for recombinant protein expression in a large number of mammalian cell lines (Lundstrom, 2003), in primary neurons (Lundstrom & Ehrengruber, 2003), in hippocampal slice cultures (Ehrengruber et al., 1999), and in vivo (Lundstrom, Richards, Pink, & Jenck, 1999). In principle, the basic method for generation of recombinant SFV particles is the same independent of further application as briefly described below. General cloning methods are applied for the subcloning of foreign genes into the SFV expression vector. After clone verification, DNA Midipreps or Maxipreps are prepared and linearized with SpeI, SapI (pSFV1 and pSFVHelper2) or NruI (pSFV2gen) prior to in vitro transcription. Generally, 5–10 μg plasmid DNA (larger quantities can be stored at -20 °C) is linearized, purified by phenol/chloroform extraction followed by ethanol precipitation. DNA pellets are resuspended in RNase-free H2O at a final concentration of 0.5 μg/μL. Alternatively, MicroSpin™ S-200 HR Columns (Amersham) can be applied according to the manufacturer’s instructions. 2.3.1 In vitro transcription High-titer virus production requires high-quality in vitro-transcribed RNA. Although manufacturers provide their own in vitro transcription kits including their SP6 buffer, it is recommended to use the optimized buffer below. To avoid precipitation due to the presence of spermidine, the in vitro transcription reactions should be set up at room temperature. Enzyme components should be added last. 5 μL (2.5 μg) linearized plasmid DNA 5 μL 10 SP6 buffer (400 mM HEPES, pH 7.4, 60 mM magnesium acetate, 20 mM spermidine) 5 μL 10 mM m7G(50 )ppp(50 )G 5 μL 50 mM DTT 5 μL rNTP mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, 5 mM rGTP) x μL RNase-free H2O to reach a final volume of 50 μL 2 μL (50 U/μL) RNase inhibitor 5 μL (20 U/μL) SP6 RNA polymerase After 1 h incubation at 37 °C 1–4 μL aliquots are loaded on a 0.8% agarose gel for RNA quality evaluation, where thick bands without smearing
336
Kenneth Lundstrom
indicate high-quality RNA. Generated RNA molecules can be directly used for electroporations or stored for weeks at -80 °C. 2.3.2 Electroporation of RNA BHK-21 cells, commonly used for production of high-titer SFV stocks, should only possess a low passage number (cultured less than 3 months) and no more than 80% confluency to provide high viability. Cells are washed once with PBS and trypsinized with 6 mL trypsin–EDTA per T175 flask for 5 min at 37 °C. Clumps are removed by resuspension and medium added to 25 mL. After centrifugation at 800 g for 5 min, the cell pellet is resuspended in a small volume (
