The authors wish to thank Dalia Barsyte-Lovejoy for taking the time to provide valuable feedback and critical comments on the manuscript and all our SGC colleagues who worked with the PRMT protein family expressed from the Baculovirus Expression Vector System.
The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].

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	<li>Guccione, E., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation,+functions+and+relevance+of+arginine+methylation.">The regulation, functions and relevance of arginine methylation.</a> Nature Reviews in Molecular Cell Biology. 20 (10), 642-657 (2019).</li><li>Thandapani, P., O'Connor, T. R., Bailey, T. L., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+RGG/RG+motif.">Defining the RGG/RG motif.</a> Molecular Cell. 50 (5), 613-623 (2013).</li><li>Lorton, B. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+beta+interferon+in+insect+cells+infected+with+a+baculovirus+expression+vector.">Production of human beta interferon in insect cells infected with a baculovirus expression vector.</a> Molecular Cell Biology. 3, 2156-2165 (1983).</li><li>Invitrogen Life Technologies. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Invitrogen, Bac-to-Bac Baculovirus expression system. , (2010).</li><li>Luckow, V. A., Lee, S. C., Barry, G. F., Olins, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+infectious+recombinant+baculoviruses+by+site-specific+transposon-mediated+insertion+of+foreign+genes+into+a+baculovirus+genome+propagated+in+Escherichia+coli.">Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.</a> Journal of Virology. 67, 4566-4579 (1993).</li><li>Irons, S. L., Chambers, A. C., Lissina, O., King, L. A., Possee, R. 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The authors declare no conflict of interest.

One of the advantages of BEVS in insect cells centers on the capability of the post-translational modification machinery to enable more complex modifications such as phosphorylation, myristoylation, and glycosylation. Together with the highly efficient folding of mammalian proteins, these modifications facilitate high amounts of modified and folded protein suitable for physiologically relevant downstream experiments16.
Here, we described detailed protocols of the BEVS emphasizing critical elements for successful expression screening of multiple constructs of PRMT proteins and large-scale PRMT protein production in the Baculovirus expression platform: 1) The use of regular, adjustable, and programmable multichannel pipettes to transfer the biological materials between 24- and 96 - well cell culture plates and blocks at the stages of bacmid DNA and virus generation; a collection of the recombinant viruses, amplification of viral volumes of the recombinant viruses and preparation of the protein expression screening blocks. 2) High performance and cost-effective transfection reagents for the generation of recombinant viruses. 3) Suspension culture of baculovirus-infected insect cells (SCBIIC) for large-scale protein production. 4) Utilization of high-fill volume 2.8 L Fernbach shake flasks to maintain Sf9 suspension culture and 2.5 L Tunair shake flasks and 5 L reagent bottles for the large-scale protein production.
Special considerations and rationale for the transformation and transfection steps. 
Although a commercial protocol recommends using 100 &#181;L of competent cells for one transformation14, the transformation efficiency of commercial DH10Bac E. coli competent cells is as high as 1 x 108 cfu/ &#181;g DNA, so we use only 4 &#181;L. This is enough for each transformant to obtain isolated white recombinant colonies for the bacmid DNA isolation. Adherent Sf9 cells in the 24-well transfection plate were seeded at a cell density of 2 x 105 /mL in 0.5 mL of Serum-Free Insect Media. This volume is enough to ensure even coverage of the working surface of the well. At the same time, it does not dilute the transfection mix too much, which enhances transfection efficiency. Transfection reagents are non-toxic to the Sf9 cells, and media exchange is not necessary. Instead of a media change, an additional 1.5 mL of media containing 10% (v/v) FBS is added into the transfection plate at 4-5 hours post-transfection time to facilitate cell growth. The transfection efficiency of both transfection reagents is high. Still, with X-tremeGene 9, the signs of infection in the transfected cells (Figure 2) appear 10-12 h earlier than with the JetPrime reagent, so we choose between these reagents depending on the working schedule of the next steps in the protocols, which provides some flexibility in the overall process.
Protein test expression screening can be set up with P2 viruses if the amount of the initial recombinant viruses, collected from the transfection plate and labeled as P1, is a limiting factor to use for the protein expression screening.
Considerations when moving from small to large culture volumes. 
Historically, it was believed that optimal cell growth requires a high air space in the suspension culture of Sf9 cell maintenance and scale-up productions. However, in 2014, it was reported that high air space in culture vessels is less critical than previously thought17. A culture vessel set up using appropriately adjusted shaking speed to the orbital throw of the shaking platform will provide sufficient oxygen transfer even in the high-fill volume suspension culture by creating and maintaining small air bubbles for a longer time. With this approach, commercially available insect cells can be cultured at a higher shaking speed within a normal range of the cells&#39; doubling time without sacrificing high cell viability.
Thus, 6 years ago, we started to increase the suspension culture volume in a shaking flask during cell maintenance and introduced a different type of culture vessel for protein production while adjusting and monitoring shaking conditions (Figure 6). To establish optimal conditions in these culture vessels, we monitored the Sf9 cell culture parameters such as&#160;cell doubling time along with cell viability, size and shape, state of aggregation, and infectability of cells.
For example, for Sf9 cell maintenance, in the 2.8 L Fernbach shake flasks, we culture 2 L instead of 0.8 L of the Sf9 suspension cells shaking at 150 rpm at 27 &#176;C and cell viability most of the time is close to 99%, with evenly shaped healthy dividing cells. For scale-up production, we infect 4 L of cells in 5 L reagent bottles shaking at high speed as 145 rpm at a lowered temperature of 25 &#176;C. The most commonly used incubators with a built-in shaking platform can hold 6 x 2.8 L shake flasks or 6 x 5 L reagent bottles, or 10 x 2.5 L Tunair shake flasks. Thus, the capacity of the one shaking platform, if we fill Fernbach shake flasks and reagent bottles to 1/3 versus to the high-fill volume of the vessels is 4.8 L versus 12 L using 2.8 L Fernbach shake flasks and 10 L versus 24 L using 5 L reagent bottles (Figure 6). Suspension cell culture maintenance and scale-up production in the culture vessels with a high-fill volume have helped us overcome limitations of the production volumes and adopt a large-scale platform. Thus, this is highly useful for labs with no access to bioreactors and/or limited space in the production pipelines.
This protocol could be readily adapted for the production and purification of protein constructs with different affinity tags by utilizing appropriate resins and modifying purification buffers as has been described in the SGC published paper6 for the Flag-tagged full-length proteins of PRMT4, 7, 9, and the His-tagged PRMT5-MEP50 complex and PRMT6. Although we describe a BEVS protocol for the PRMT family of proteins, the same approach can be applied to any other protein family.

Protein arginine methyltransferases (PRMTs) methylate arginine residues in a monomethyl or symmetric/asymmetric dimethyl fashion. The repetitive RG/RGG/GRG sequences are highly preferred by most PRMTs and are found in a wide variety of proteins1,2. Arginine methylated proteins such as histones or transcription factors and splicing factors regulate transcription, splicing, and chromatin structure3,4. Increasing knowledge of diverse regulation of substrate and cofactor utilization, turnover, and kinetics of PRMTs, as well as generation of selective inhibitors, have shed mechanistic light on these enzymes and their complexes5,6. However, not all PRMT family members are studied to the same extent; for example, PRMT9 was only recently discovered to be a member of the PRMT family1. Structure and enzyme function studies for these proteins require sufficient, often milligram, quantities of recombinant protein to be available.
The Escherichia coli (E. coli) prokaryotic expression system is usually the first choice for expression screening utilizing multiple constructs for a given protein7,8,9. However, E. coli-based expression does not always result in sufficient quantities of PRMT proteins in their active forms, as we have noted in particular for PRMT5 and PRMT7 (see below). Thus, PRMTs that failed to express in E. coli or needed to be produced by the eukaryotic expression machinery were subcloned into vectors appropriate for the expression screening in the alternative baculovirus expression vector system (BEVS). While E. coli expressed samples of PRMT1, PRMT3 and PRMT8 have been utilized extensively for in vitro assays and crystallography, other PRMTs such as PRMT5, which requires MEP50 binding partner of its dual methyltransferase domain, and PRMTs such as PRMT7 and 9, necessitate insect cell expression to obtain sufficient quantities of active protein. Overall, the standardized medium-throughput methyltransferase assays for PRMT4, 5, 6, 7, and 9 have utilized the BEVS in insect cells6. The baculovirus expression vector system (BEVS) is a versatile platform to produce recombinant proteins requiring the eukaryotic expression machinery that enables post-translational modifications essential for biochemical, biophysical, and structural studies10,11,12. Several BEVSs have become commercially available since the first reported use of baculoviruses in 1983 for protein expression13. Most of these protocols employ different strategies for the transfer of the expression plasmid into insect cells. These include Bac-to-Bac, flashBAC, BaculoGOLD Bright, BacVector-3000, BacMagic, BacPAK, etc. Our protocol is based on the most commonly used system in BEVS, the Bac-to-Bac system14, which is designed to transfer the gene/cDNA encoding the protein of interest (POI, here the PRMTs) into the baculovirus genome maintained in a specialized strain of E. coli via site-specific transposition15.
Briefly, the plasmid transfer vector containing the gene of interest was transformed into DH10Bac E. coli competent cells to generate recombinant viral bacmid DNA. Adherent Sf9 cells were then transfected with bacmid DNA. Four to five days after transfection, initial recombinant baculoviruses secreted into the cell culture medium were recovered and labeled as the P1 virus. The P1 baculovirus stocks were then used for virus amplification (i.e., generation of P2 baculovirus stocks) and protein expression screening. Based on the expression screening results, P2 viruses for the best expression construct of the protein were identified and used to generate suspension cultures of baculovirus-infected insect cells (SCBIIS) for the large-scale protein production. Here, we describe our detailed protocols and describe the rationale behind our reagent and culture vessel choices to support our strategy of developing a more time-efficient, cost-efficient, and scalable methodology to obtain sufficient quantities of desired recombinant proteins.

<table><tbody><tr><td>2.8L Nalgene&amp;nbsp; Fernbach Culture Flask, Polycarbonate,</td><td>Nalgene</td><td>29171-854</td><td>For large scale maintenance of suspension culture of Sf9 cells</td></tr><tr><td>24-Well Blocks RB</td><td>Qiagen</td><td>19583</td><td>For incubation of&amp;nbsp; 4ml of suspension&amp;nbsp; Sf9 cells&amp;nbsp; for protein&amp;nbsp; expression screening</td></tr><tr><td>4-20 Criterion TGX Gel 26W 15 ul</td><td>Biorad</td><td>5671095</td><td>For SDS-PAGe analysis of the purified proteins</td></tr><tr><td>50ml Reagent Reservoir</td><td>Celltreat Scientific Products</td><td>229290</td><td>&amp;nbsp;Reservoir used for diluted transfection reagent&amp;nbsp; and Sf9 cells suspension</td></tr><tr><td>96-well cap mat, for use with square well, 2 mL</td><td>Greiner Bio-One</td><td>381080</td><td>Used to cover 96 well block</td></tr><tr><td>96 well PCR plate</td><td>Eppendorf</td><td>30129300</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>214010</td><td>For LB-agar selection paltes</td></tr><tr><td>Airpore Tape Sheets</td><td>Qiagen</td><td>19571</td><td>To cover 24 well blocks for protein expression screening</td></tr><tr><td>Allega X-15R Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>Antibiotic Antimycotic (100x)</td><td>Gibco</td><td>15240112</td><td></td></tr><tr><td>Bacmid DNA</td><td>in-house</td><td>non-catalog item</td><td>Bacmid DNA for baculovirus production</td></tr><tr><td>Bluo-Gal, 1g</td><td>Thermo Fisher</td><td>15519028</td><td></td></tr><tr><td>Beckman JLA 8.1000</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Plates, 24-Well, with lid, flat bottom, sterile</td><td>Eppendorf</td><td>30722116</td><td>Tissue&amp;nbsp; culture treated plate</td></tr><tr><td>Cell Resuspension Solution 0.5&amp;nbsp;L</td><td>Millipore Sigma</td><td>LSKCRS500</td><td>For Bacmid DNA extraction</td></tr><tr><td>Cell&amp;nbsp;Lysis Solution 0.5&amp;nbsp;L</td><td>Millipore Sigma</td><td>LSKCLS500</td><td>For Bacmid DNA extraction</td></tr><tr><td>CELLSTAR&amp;nbsp; Tissue Culture Plates, 96 well</td><td>Greiner Bio-One</td><td>655180</td><td>For transfection mix</td></tr><tr><td>ClipTip 1250, filter reload, sterile</td><td>ThermoFisher Scientific</td><td>94420818</td><td>Tips for&amp;nbsp; programmable and adjustable multichannel pipette</td></tr><tr><td>dNTP Mix (25 mM each)</td><td>Thermo Fisher Scientific</td><td>R1121</td><td></td></tr><tr><td>E1-ClipTip&amp;nbsp; Electronic Adjustable Tip Spacing Multichannel Equalizer Pipette, 15 to 1250 &amp;mu;L</td><td>ThermoFisher Scientific</td><td>4672090BT</td><td>Programmable and adjustable multichannel pipette</td></tr><tr><td>E4&amp;nbsp; XLS adjustable spacer 6-channel pipette, 20-300 &amp;mu;L</td><td>Ranin</td><td>LTS EA6-300XLS</td><td>Ranin adjustable multichannel pipette</td></tr><tr><td>Filter microplate, 96-well, polypropylene, with 25 &amp;micro;m ultra high molecular weight polyethylene membrane</td><td>Agilent</td><td>201005-100</td><td>For protein purification in expression screening</td></tr><tr><td>Full-Baffle Flask Kit Tunair, 2.5L</td><td>IBI Scientific</td><td>SS-6003C</td><td>For large scale protein production in suspension culture of SF9 cells</td></tr><tr><td>Gentamicin 10x10ml</td><td>BioShop</td><td>15750078</td><td></td></tr><tr><td>Heat Inactivated Fetal Bovine Serum</td><td>Wisent Biocenter</td><td>080-450</td><td></td></tr><tr><td>I-Max Insect Media W/ L-Glutamine, 1 L</td><td>Wisent Biocenter</td><td>301-045-LL</td><td>Serum free insect cells growth medium</td></tr><tr><td>InstantBlue, Ultrafast Protein Stain</td><td>Expedeon Protein Solutions</td><td>ISB1L-1L</td><td>For protein gel (SDS-PAGE) staining</td></tr><tr><td>Iptg, Ultra Pure, Dioxane Free, Min 99.5%</td><td>BioShop</td><td>IPT001.100</td><td></td></tr><tr><td>JetPRIME Transfection Reagent&amp;nbsp; Provided with&amp;nbsp; jetPRIME buffer</td><td>POLYPLUS TRANSFECTION Inc</td><td>114-01</td><td>For Sf9 cells transfection to generate baculovirus</td></tr><tr><td>Kanamycin Monosulfate</td><td>BioShop</td><td>KAN201.100</td><td></td></tr><tr><td>Lb Broth (Lennox), Powder Microbial Gro&amp;amp;</td><td>Sigma</td><td>L3022-1KG</td><td></td></tr><tr><td>Masterblock 96 deep well 2.4mL</td><td>Greiner Bio-One</td><td>780285-FD</td><td>Used in the transformation and expression screening procedures</td></tr><tr><td>Max Efficiency DH10Bac Competent Cells , 0.5ml</td><td>Thermo Fisher</td><td>10361012</td><td>Competent cells for bacmid DNA generation</td></tr><tr><td>mLINE 12-Channel Pipette, adjustable 30 - 300 uL</td><td>Sartorius</td><td>Sartorius 725240</td><td>12 channel pipette</td></tr><tr><td>Neutralization Solution, 0.5&amp;nbsp;L</td><td>Millipore Sigma</td><td>LSKNS0500</td><td>For bacmid DNA extraction</td></tr><tr><td>New Brunswick&amp;nbsp; Innova 44R, 120V, orbit 2.5 cm (1 in)</td><td>Eppendorf</td><td>M1282-0004</td><td>Shaker incubator for incubaion of suspension of Sf9 cells</td></tr><tr><td>Ni-NTA Agarose</td><td>Qiagen</td><td>30250</td><td>For protein purification</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>LS15140122</td><td></td></tr><tr><td>PYREX&amp;nbsp; Delong Shaker Erlenmeyer Flask with Baffles, Corning&amp;nbsp; 500ml</td><td>Pyrex</td><td>4444-500</td><td>For&amp;nbsp; suspension culture of Sf9 cells</td></tr><tr><td>PYREX Delong Shaker Erlenmeyer Flask with Baffles, Corning&amp;nbsp; 125ml</td><td>Pyrex</td><td>4444-125</td><td>For suspension culture of Sf9 cells</td></tr><tr><td>PYREX Delong Shaker Erlenmeyer Flask with Baffles, Corning&amp;nbsp; 250ml</td><td>Pyrex</td><td>4444-250</td><td>For suspension culture of Sf9 cells</td></tr><tr><td>RedSafe&amp;nbsp;Nucleic&amp;nbsp;Acid&amp;nbsp;Staining&amp;nbsp;Solution</td><td>Froggabio</td><td>21141</td><td></td></tr><tr><td>RNase A, 0.9&amp;nbsp;mL</td><td>Millipore Sigma</td><td>LSKPMRN30</td><td>For suspension buffer for bacmid DNA extraction</td></tr><tr><td>Roll &amp;amp; Grow Spherical Glass Plating Beads</td><td>MP Biomedicals</td><td>115000550</td><td>For&amp;nbsp; spread&amp;nbsp; of bacterial&amp;nbsp; cells across the surface of an agar plate.</td></tr><tr><td>RT-LTS-A-300&amp;mu;L-768/8 (tips)</td><td>Ranin</td><td>30389253</td><td>Tips for ranin multichannel pipette</td></tr><tr><td>S.O.C. Medium</td><td>Thermo Fisher Scientific</td><td>15544034</td><td></td></tr><tr><td>Serum, Cell Culture, Fetal Bovine Serum (Fbs), Hyclone, Characterized Canadian</td><td>cytivalifesciences</td><td>SH3039602</td><td>Addition to the serum free medim for&amp;nbsp; the transfected cells</td></tr><tr><td>Sf9 cells</td><td>Thermo Fisher</td><td>12659017</td><td>Insect cells</td></tr><tr><td>Sfx-Insect Cell Culture Media</td><td>Cytiva (Formerly GE Healthcare Life Sciences)</td><td>SH3027802</td><td>Serum free insect cells growth medium</td></tr><tr><td>Tape Pad</td><td>Qiagen</td><td>19570</td><td>Tape Pad</td></tr><tr><td>Taq DNA Polymerase with ThermoPol Buffer - 2,000 units</td><td>New England Biolabs</td><td>M0267L</td><td></td></tr><tr><td>Tetracycline Hcl</td><td>BioShop</td><td>TET701.10</td><td></td></tr><tr><td>Trypan Blue 0.4% Solution</td><td>Gibco</td><td>15250061</td><td>For&amp;nbsp; assessment of cell viability</td></tr><tr><td>VITLAB&amp;nbsp; Reagent Bottles, PP with Screw Caps, PP, BrandTech, 5L</td><td>VITLAB</td><td>V100889</td><td>For large scale protein production in suspension culture of Sf9 cells</td></tr><tr><td>VWR&amp;nbsp; Digital Mini Incubator</td><td>VWR</td><td>10055-006</td><td>Incubator for adherent Sf9 cells</td></tr><tr><td>VWR&amp;nbsp; Incubating Microplate Shaker</td><td>VWR</td><td>97043-606</td><td>Incubator for suspension culture of Sf9 cells in 24 well blocks</td></tr><tr><td>VWR Petri Dishes</td><td>VWR</td><td>CA73370-037</td><td></td></tr><tr><td>X-tremeGene 9 DNA&amp;nbsp; Transfection Reagent&amp;nbsp; 1.0 M</td><td>Roche</td><td>6365787001</td><td>For Sf9 cells transfection to generate recombinant baculovirus</td></tr></tbody></table>

production of recombinant prmt proteins using the baculovirus expression vector system

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

An overview of the BEVS protocol is outlined in Figure 1. Multiple expression constructs of PRMTs, including full-length, domains, and truncated fragments, were generated at the Structural Genomics Consortium (SGC, Toronto) according to in-house strategies with an attempt to increase the success rate for identifying soluble and stable proteins with a relatively high expression level7,9. Interested readers are encouraged to review the SGC&#39;s definitions and methodology of designing a &#34;fragment&#34; as the segment of the gene sequence incorporated into an expression clone, &#34;domain&#34; as a PFAM-annotated structural domain, and &#34;construct&#34; as the fragment cloned in an expression vector, all of which have been described in detail in an earlier publication7. Expression constructs of PRMTs presented in this protocol are for the production of the polyhistidine-tagged proteins cloned into the pFBOH-MHL vector, which is a derivative of the pFastBac1 vector. In Figure 4, we present SDS-PAGE analysis of the His-tagged soluble constructs of PRMT1, 2, 4-9 purified from pellets collected after 4 mL of production in Sf9 cells (step 3.1.4). Full-length (FL) PRMT1 and PRMT9 are not presented in this gel, since FL PRMT1 has been produced from E. coli, and FL PRMT9 produced from BEVS has been purified by Flag-tag6. The truncated constructs of PRMT1, FL PRMT4, and all the PRMT8 constructs show a relatively high yield, but protein eluates contain fractions of co-purified contaminants. These constructs require further optimization of the purification protocols. Additional approaches are thus required to improve the purity of these proteins from scale-up productions, such as a reduction in the amount of nickel beads at the stage of the incubation with a clarified lysate; an increase of the imidazole concentrations in the wash buffers; cleavage of the His-tag with TEV protease, followed by application on a Ni-affinity resin; and, additional purification steps such as size-exclusion and ion-exchange chromatography. The constructs of PRMT2 show significantly lower yield compared to other proteins and full-length PRMT2 protein accompanied by a strong contaminant band. Scale-up production and two steps of purifications such as IMAC and size-exclusion confirmed a low expression level for this construct along with the persistent presence of the co-purifying contaminant for the FL protein. Pure proteins have been obtained for the PRMT5 complex produced and purified with its obligate binding partner, MEP50. The truncated construct of PRMT9 has almost two or three-fold lower expression level, close to 1.5 mg/L, as compared to other PRMTs. Nevertheless, the recombinant viral stocks of this construct have been used for scale-up production, diffracting crystals were obtained, and the structure was solved for this protein along with PRMT4, 6, and 7 (Figure 5).
For the scale-up productions, the corresponding P2 viruses were used to infect the suspension culture of Sf9 insect cells. This step generates 50/100/200 mL of baculovirus-infected cells containing infected cells and P3 viruses in the supernatant. For the large-scale protein production, 2 L of Sf9 cells were cultured in each of 2.8 L Fernbach shake flasks at 150 rpm, 27 &#176;C (Figure 6). On the day of production, 2 L of Sf9 cells (cell viability &#62; 97%) in 2.5 L Tunair shake flasks or 4 L in 5 L reagent bottles were diluted to a cell density of 4 x 106/mL. These cells were infected directly with 10-12 mL/L of suspension culture of baculovirus-infected insect cells and incubated at a lowered temperature of 25 &#176;C, at 145 rpm. Infection of the production batch directly with a suspension culture of baculovirus-infected insect cells significantly reduced laborious and time-consuming steps in virus volume amplification, excluding the extra handling of the infected cells, and avoided reduction in titer and virus degradation. SF9 cell culture maintenance and scale-up production have been done in the culture vessels with a high-fill volume to adopt a large-scale protein production in the one batch (Figure 6).
Full-length PRMT 4, 5 (in complex with MEP50), 6, 7, and 9 proteins produced from the Baculovirus mediated production platform have been used for kinetic characterization and inhibitor compound screening at the SGC6. Crystal structures were solved and deposited into the Protein Data Bank (PDB) for the full-length or truncated forms of the proteins PRMT 4, 6, 7, and 9 with various chemical probes and inhibitors. Expression plasmids for these PRMTs were deposited to the Addgene plasmid repository (Addgene is a distributing partner of the SGC, https://www.addgene.org/) and are available to the research community (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62510/62510fig01.jpg" /> 
Figure 1: Schematic overview of the steps of the baculovirus expression process <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62510/62510fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62510/62510fig02.jpg" /> 
Figure 2: Baculovirus Infected and uninfected Sf9 cells. Signs of infection are structural changes in the insect cells, such as a 25-50% increase in the cell diameter, enlarged cell nuclei, uniformly rounded shape, loss of proliferation, and adherence to the culture dish surface, as well as a decrease in cell viability. White scale bar 200 &#181;m. The signs of infection presented here are the same for the transfected cells using both transfection reagents, JetPrime and X-tremeGene 9. The particular example shown is for the JetPrime transfection reagent. (A) Uninfected Sf9 cells as a control. (B) Baculovirus-infected Sf9 cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62510/62510fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62510/62510fig03.jpg" /> 
Figure 3:&#160;Binding plate assembly for quick purification of test expression proteins. Please see text for details, steps 3.2.1-2 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62510/62510fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62510/62510fig04.jpg" /> 
Figure 4:&#160;Protein expression screening results. Protein expression screening results of the baculovirus mediated protein production in 4 mL of Sf9 suspension culture infected with corresponding P1 recombinant viruses for different PRMTs and the PRMT5-MEP50 complex. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62510/62510fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62510/62510fig05.jpg" /> 
Figure 5:&#160;Summary of expression constructs for PRMT4, 6, 7, and 9 used for crystal structure studies at the Structural Genomics Consortium, Toronto (SGC). The crystal structures were solved and deposited into the Protein Data Bank (PDB) for the full length or truncated forms of proteins PRMT 4, 6, 7, and 9 with various chemical probes and inhibitors. Expression plasmids for these PRMTs were deposited to the Addgene plasmid repository and are available to the research community (Addgene is a distributing partner of the SGC, https://www.addgene.org/).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62510/62510fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62510/62510fig06.jpg" /> 
Figure 6:&#160;Sf9 insect cell maintenance and protein production in the different culture vessels: (A) 2.8 L Fernbach flask for cell maintenance and protein production. The use of the 72% fill-volume increases the throughput rate by 2.5-fold in one shaking platform. (B) Tunair shake flasks (only 9 flasks out of 10 are presented in this picture) and reagent bottles with an 80% fill-volume drastically increase the shaking platform&#39;s production capacity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62510/62510fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System at JoVE.com

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

production-recombinant-prmt-proteins-using-baculovirus-expression

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

7.2K Views.  University of Toronto. The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform. 

Video: Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Genetics

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Biology

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...