Name: production of recombinant prmt proteins using the baculovirus expression vector system
Uploaded: 2021-07-17T14:00:00
Duration: 8 min 57 s
Description: Watch this Scientific Journal Video about Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System at JoVE.com

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].

<ol>
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A., Condreay, J. P., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+as+versatile+vectors+for+protein+expression+in+insect+and+mammalian+cells.">Baculovirus as versatile vectors for protein expression in insect and mammalian cells.</a> Nature Biotechnology. 23, 567-575 (2005).</li><li>Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-insect+cell+expression+systems.">Baculovirus-insect cell expression systems.</a> Methods in Enzymology. 463, 191-222 (2009).</li><li>Shrestha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods in Molecular Biology. 439, 269-289 (2008).</li><li>Smith, G. E., Summers, M. D., Fraser, M. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.8L Nalgene&#160; 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&#160; 4ml of suspension&#160; Sf9 cells&#160; for protein&#160; 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>&#160;Reservoir used for diluted transfection reagent&#160; 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&#160; culture treated plate</td>
		</tr>
		<tr>
			<td>Cell Resuspension Solution 0.5&#160;L</td>
			<td>Millipore Sigma</td>
			<td>LSKCRS500</td>
			<td>For Bacmid DNA extraction</td>
		</tr>
		<tr>
			<td>Cell&#160;Lysis Solution 0.5&#160;L</td>
			<td>Millipore Sigma</td>
			<td>LSKCLS500</td>
			<td>For Bacmid DNA extraction</td>
		</tr>
		<tr>
			<td>CELLSTAR&#160; 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&#160; 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&#160; Electronic Adjustable Tip Spacing Multichannel Equalizer Pipette, 15 to 1250 &#956;L</td>
			<td>ThermoFisher Scientific</td>
			<td>4672090BT</td>
			<td>Programmable and adjustable multichannel pipette</td>
		</tr>
		<tr>
			<td>E4&#160; XLS adjustable spacer 6-channel pipette, 20-300 &#956;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 &#181;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&#160; Provided with&#160; 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&#38;</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&#160;L</td>
			<td>Millipore Sigma</td>
			<td>LSKNS0500</td>
			<td>For bacmid DNA extraction</td>
		</tr>
		<tr>
			<td>New Brunswick&#160; 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&#160; Delong Shaker Erlenmeyer Flask with Baffles, Corning&#160; 500ml</td>
			<td>Pyrex</td>
			<td>4444-500</td>
			<td>For&#160; suspension culture of Sf9 cells</td>
		</tr>
		<tr>
			<td>PYREX Delong Shaker Erlenmeyer Flask with Baffles, Corning&#160; 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&#160; 250ml</td>
			<td>Pyrex</td>
			<td>4444-250</td>
			<td>For suspension culture of Sf9 cells</td>
		</tr>
		<tr>
			<td>RedSafe&#160;Nucleic&#160;Acid&#160;Staining&#160;Solution</td>
			<td>Froggabio</td>
			<td>21141</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A, 0.9&#160;mL</td>
			<td>Millipore Sigma</td>
			<td>LSKPMRN30</td>
			<td>For suspension buffer for bacmid DNA extraction</td>
		</tr>
		<tr>
			<td>Roll &#38; Grow Spherical Glass Plating Beads</td>
			<td>MP Biomedicals</td>
			<td>115000550</td>
			<td>For&#160; spread&#160; of bacterial&#160; cells across the surface of an agar plate.</td>
		</tr>
		<tr>
			<td>RT-LTS-A-300&#956;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&#160; 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&#160; assessment of cell viability</td>
		</tr>
		<tr>
			<td>VITLAB&#160; 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&#160; Digital Mini Incubator</td>
			<td>VWR</td>
			<td>10055-006</td>
			<td>Incubator for adherent Sf9 cells</td>
		</tr>
		<tr>
			<td>VWR&#160; 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&#160; Transfection Reagent&#160; 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/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/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/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/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/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/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. ...

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