Name: production and purification of baculovirus for gene therapy application
Uploaded: 2018-04-09T13:30:00.000+00:00
Duration: 6 min 11 s
Description: Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

This work is supported in part by the Start-Up funding from Cincinnati Children's Research Foundation (CCRF) to M.N. and Innovative Core Grant (ICG) supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1 TR001425. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

See Figure 1 for an illustration summarizing the protocol.
1. Purification of Baculovirus Plasmid DNA
<ol>
	<li>Grow bacterial culture containing baculoviral plasmid DNA (bacmid)14 in 200 mL of LB broth with antibiotics; kanamycin (50 &#181;g/mL), tetracycline (10 &#181;g/mL), and gentamycin (7 &#181;g/mL), for 16 h at 37 &#176;C on an orbital shaker setting at 300 rpm.</li>
	<li>Purify the bacmid DNA from the bacterial culture using standard alkaline lysis protocol15, and dissolve the bacmid in TE buffer.</li>
</ol>
2. Production of Baculovirus
<ol>
	<li>Culture Sf9 cells in an orbital shaker incubator at 135 rpm and 28 &#176;C in a polycarbonate Erlenmeyer flask containing serum-free insect culture medium1,2,3. 
	Note: If Sf9 cells are thawed from frozen stock, allow at least two weeks for the cells to recover and enter the exponential phase of growth.</li>
	<li>Count the Sf9 cells harvested at exponential phase of growth with a hemocytometer after staining with Trypan blue. Seed 1 x 106 viable Sf9 cells per well in a 6-well tissue culture-treated plate in 2 mL of medium. Allow the cell to attach for 1 h at 28 &#176;C in an incubator.</li>
	<li>Replace the growth medium with 1 mL of serum-free unsupplemented Grace&#39;s insect culture medium.</li>
	<li>Transfect the bacmid DNA into the Sf9 cells using insect cell transfection reagent.
	<ol>
		<li>Mix 8 &#181;L insect cell transfection reagent with 100 &#181;L of Grace&#39;s Insect medium.</li>
		<li>Dilute 2 &#181;g of bacmid DNA into 100 &#181;L of unsupplemented insect medium, and mix gently by vortexing.</li>
		<li>Combine the diluted DNA with the diluted insect cell transfection reagent, and mix gently. Incubate for 15 to 30 min at room temperature.</li>
		<li>Add the DNA-lipid mixture dropwise onto the cells. Incubate at 28 &#176;C in an incubator for approximately 5 h.</li>
	</ol>
	</li>
	<li>Remove the transfection mixture from the cells and wash the cells with 2 mL of PBS.</li>
	<li>Add 2 mL of insect culture medium and continue to culture at 28 &#176;C in an incubator without changing the media. 
	Note: If the bacmid contains a fluorescent protein, its expression can be detected in cells 48 h post-transfection. Baculovirus is produced by transfected Sf9 cells and secreted into the culture medium which subsequently infects the untransfected cells. The entire cell population becomes infected with baculovirus in 5 days and shows signs of viral infection, also called cytopathic effect. These include increased cell diameter, vacuoles/granules in the cytoplasm, cessation of cell growth and dead or lysed cells are present in cell culture. However, if the transfection or infection efficiency is not optimal, the culture may not show an obvious cytopathic effect. The cytopathic effect can be visualized with an inverted phase microscope at 200 - 400X magnification. Baculovirus infected cell can be detected by staining with anti-gp64 antibody11.</li>
	<li>Harvest baculovirus supernatant 5 days after transfection and label as the P1 virus stock.</li>
	<li>Store the baculovirus supernatant, which is light sensitive, in the dark with 0.5% bovine serum albumin (BSA) in PBS. Store at 4 &#176;C for the short-term (up to 90 days) or at -80 &#176;C for the long-term.</li>
	<li>Seed 6 &#215; 106 Sf9 cells in a 10-cm tissue culture-treated plate in 10 mL of insect culture medium. Add 1 mL of initial baculovirus supernatant (P1) to the cells.</li>
	<li>Harvest baculovirus supernatant (P2) at 5th day post-infection.</li>
	<li>Seed 50 mL of Sf9 cells (3 x 106 cells/mL) in suspension culture in insect culture medium in a 250-mL polycarbonate Erlenmeyer flask and place the flask in an orbital shaker incubator. Add 2 mL of P2 stock to the cells and shake at 135 rpm while maintaining a constant temperature of 28 &#176;C. 
	Note: Three or four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high-titer baculovirus production.</li>
	<li>Sequentially amplify the baculovirus supernatants until the desired volume is obtained (P3, P4, &#8230;.) by increasing 10 to 50-fold volume of cell culture in each subsequent infection. 
	Note: P3 is the 3rd round of infection in which 500 mL to 1 L of Sf9 cell culture can be infected with 10 to 20 mL of P2 baculovirus supernatant; P4 is the 4th round of infection in which 5 to 10 L of Sf9 cell culture can be infected with 100 to 200 mL of P3 baculovirus supernatant, and so on. Typically, Sf9 cells are seeded at 3 &#215; 106 cells/mL in serum-free insect culture media.</li>
	<li>Centrifuge the baculovirus supernatant at 1,000 x g for 30 min at 4 &#176;C to remove&#160;cellular debris and treat the baculovirus supernatant with 50 units/mL nuclease (250 units/&#181;L of stock) for 2 h at 37 &#176;C to digest the genomic DNA and RNA. Filter the supernatants through a 0.45 &#956;m filtration unit.</li>
</ol>
3. Preparation of the Chromatography System
<ol>
	<li>Prepare the chromatography system by sequentially rinsing the sample and buffer lines each with sterile water, 1 N sodium hydroxide, water, and wash buffer (20 mM sodium phosphate buffer containing 150 mM sodium chloride, pH 7.0) at a linear flow rate of 50 mL/min.</li>
	<li>Prepare a heparin 50 &#181;m column (10 &#215; 10 cm, 7.9 mL of column volume (CV)) by sequentially running column cleaning buffer (5 CV sterile 20 mM sodium phosphate buffer containing 2 M sodium chloride, pH 7.0) and 5 CV wash buffer at a linear flow rate of 7 mL/min.</li>
</ol>
4. Purification of Baculovirus Vector
<ol>
	<li>Set up the chromatography system as follows:
	<ol>
		<li>Use the sample loading inlet port for loading the baculovirus supernatant.</li>
		<li>Use the buffer inlet port A1 for loading the wash buffer. Prepare a bottle with at least 500 mL of wash buffer and place the wash buffer line into the bottle.</li>
		<li>Use the buffer inlet port A2 for loading the elution buffer (20 mM sodium phosphate with 1.5 M sodium chloride, pH 8.0). Prepare a bottle with at least 500 mL of elution buffer and place the elution buffer line into the bottle.</li>
		<li>Insert several 50 mL conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluted baculovirus.</li>
	</ol>
	</li>
	<li>Equilibrate the heparin column with five 7.9 mL column volumes (CVs) of wash buffer (40 mL) at a linear flow rate of 7.0 mL/min.</li>
	<li>Load 250 mL of baculovirus supernatant onto the heparin column using the sample pump (inlet sample port) of the chromatography system at a linear flow rate of 2.0 mL/min.</li>
	<li>Run 10 CVs (80 mL) of wash buffer through heparin column at a linear flow rate of 2.0 mL/min until the ultraviolet (UV) absorbance curve (280 nm) has returned to baseline and becomes stable.</li>
	<li>Elute the baculovirus particles from the 7.9 mL heparin column with 5 CVs (40 mL) of elution buffer at a linear flow rate of 4.0 mL/min. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.</li>
	<li>Post-elution, immediately dilute the eluted baculovirus supernatant 10-fold using 20 mM sodium phosphate buffer in water to prevent inactivation of baculovirus particles from osmotic shock during subsequent centrifugation.</li>
	<li>Store 100 &#181;L of each of the fractions, including the column flow-through collected during loading of the baculovirus supernatant, the wash buffer, and the eluate. Infect these baculovirus fractions to HT1080 to evaluate the purification process (See section 6).</li>
	<li>Clean the column with&#160;column cleaning buffer and&#160;wash buffer. Finally, rinse the column with 5 CV&#160;sterile 20% ethanol in water at a linear flow rate of 7.0 mL/min and store at 4 &#176;C.</li>
	<li>Clean the chromatography system with water, 1 N sodium hydroxide, again with water, and&#160;20% ethanol in water at a linear flow rate of 50.0 mL/min and store the system in 20% ethanol.</li>
</ol>
5. Concentration of Baculovirus
<ol>
	<li>Pre-sterilize centrifuge tubes using an autoclave. Add an equal volume of baculovirus supernatant to each ultracentrifuge tubes in a tissue culture hood. Measure the tube weight by a balance and adjust the weight of the contents of the ultracentrifuge tubes with sterile PBS.</li>
	<li>Place each of the ultracentrifuge tubes in opposing bucket of the SW 32 Ti rotor.</li>
	<li>Run the ultracentrifuge at 80,000 &#215; g for 90 min at 4 &#176;C.</li>
	<li>Open the ultracentrifuge tubes in a tissue culture hood and aseptically aspirate the liquid without removing the baculovirus pellet.</li>
	<li>Resuspend the pellet of each tube by vigorously pipetting up and down with 200 &#181;L PBS containing 0.5% (w/vol) BSA.</li>
	<li>Transfer the concentrated baculovirus to cryovials (50 to 100 &#181;L per vial) and store at -80 &#176;C.</li>
</ol>
6. Titration of Baculovirus
<ol>
	<li>The day before infection, count HT1080 cells using a hemocytometer after staining cells with Trypan blue. Seed 1 x 105 HT1080 cells per well in a 6-well plate in 2 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) containing 10% fetal bovine serum (FBS). Seed several additional wells to be able to determine the exact number of cells present at the time of infection. Culture the cells in an incubator at 37 &#176;C and 5% CO2. 
	Note: HT1080 cells are used for titration as they express a higher level of heparan sulfate as compared to the other cell lines16. Since HT1080 cells are adherent, titration is simpler and less time consuming as compared to using the traditional Sf9-based titration.</li>
	<li>On the day of infection, determine the number of cells per well by counting cells from wells. Infect the cells by adding the diluted original baculovirus which had been set aside prior to column purification, and with samples from the column flow-through, wash, and elution fractions. For each sample, determine the dilution and volume empirically based on infectious baculovirus particles, and infect each well in triplicate.</li>
	<li>Two days after infection, analyze the cells for the expression of the gene of interest. Cells can be analyzed using a flow cytometer if they express a fluorescent protein (e.g. GFP)17.</li>
	<li>Calculate the titers (infectious unit per mL) based on the number of cells present at the time of infection, the dilution factor, and percentages of fluorescent protein in cells using the formula: (Number of cells during infection &#215; percentage of fluorescent protein positive cells &#215; dilution factor)/volume of baculovirus in mL. 
	Note: For example, if total number of cells per well at the time of infection is 1.2 &#215; 105, the percentage of fluorescent protein is 5%, the dilution factor is 100, and the volume of diluted baculovirus added is 10 &#181;L, the titer is 6 &#215; 107 infectious units (IU)/mL.</li>
</ol>

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The authors declare no conflict of interest.

The protocol presented here describes the production of baculovirus in Sf9 cells in suspension culture and purification of baculovirus using a heparin affinity chromatography. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus. The protocol provided here shows a significantly improved recovery of baculovirus particles as compared to recoveries achieved by others9.
Due to broad host range and tissue tropism, several studies were conducted in the central nervous system, liver, eye, ovary, prostate, and testis with baculovirus-mediated gene delivery5,6,7,8. Baculovirus vectors containing therapeutic genes, such as suicide genes, tumor suppressor genes, and genes encoding tumor-specific antigens have successfully been conducted in preclinical animal models18,19. Although baculovirus can transduce human cells, it can only be propagated in insect cells and, as a result, does not pose a risk to human recipients.
Baculovirus is produced in insect cells by transient transfection of bacmid DNA. The quality of bacmid is critical for the production of high-titer baculovirus. Typically, freshly prepared bacmid performs better than those are stored in the refrigerator for long period of time. Actively growing Sf9 cells are transfected efficiently which produce high-titer baculovirus. During the amplification phase, it is important to optimize the cell concentration in the suspension culture and the volume of baculovirus supernatant used for infection. If the ratio of baculovirus particles to cells is not optimal, the yield of baculovirus may be lower than the expected (personal observation, MN).
While loading the baculovirus supernatant onto the heparin column, it is important to check the flow-through for virus particles that may be passing through the column. If that occurs, a lower column run speed may be necessary or smaller volume of supernatant should be loaded onto the column. Most of the contaminants present in the baculovirus supernatants are washed off during the washing step of the chromatography run. We evaluated the purity of baculovirus by silver staining of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and electron microscopic study and found that our purified baculoviruses are good quality12.
We have carefully optimized the binding conditions and found that the POROS heparin medium is superior to other heparin media (e.g. HiTrap or Capto heparin) in its ability to capture baculovirus particles (personal observation). The ability of heparin to bind baculovirus at higher sample speed and capacity is important to minimize downstream processing time for the large-scale manufacturing process. Besides baculovirus, heparin medium also binds other contaminating proteins that have an affinity for heparin. Most of the low molecular weight contamination can be eliminated by including a tangential flow filtration (TFF) step downstream of the heparin chromatography run. TFF is also used as an alternative to centrifugation to concentrate the product20.
This protocol can be used for the purification of other viruses and proteins that have an affinity for heparin. However, modifications to the wash and elution buffer composition and the flow rate may be required.
Recombinant protein production works well using unpurified baculovirus supernatants directly. Therefore, crude baculovirus can be used for recombinant protein production. However, if baculoviruses are used as a vector for in vivo gene transfer, or as a vaccine, additional purification steps such as chromatography, gel filtration and/or ultracentrifugation are necessary to obtain pure and concentrated baculovirus particles and minimize producer cell- and culture media-derived impurities to avoid toxicity, inflammation, and an immune response.
In conclusion, we have described a simple protocol for the production and purification of baculovirus which can be scaled-up for the manufacturing recombinant proteins, vaccines, and gene therapy vectors.

Baculovirus is primarily used for the production of recombinant proteins and vaccines in lepidopteran Spodoptera fugiperda (Sf)9 insect cells by using recombinant Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV)1,2,3,4. More recently, it is emerging as a promising vector for gene therapy application5. It is known to have a broad host and tissue tropism, infects both quiescent and proliferating cells, is non-pathogenic, and does not integrate into the host chromosome4,5,6. Moreover, baculovirus can be produced in serum-free suspension culture which is scalable and allows for closed system processing for future clinical production1.
The purity of baculovirus particles is important for achieving effective transduction while minimizing cytotoxicity7,8,9. Baculovirus can be concentrated by ultracentrifugation or tangential flow filtration (TFF) with limited impact on its infectivity. However, these procedures not only concentrate virus particles but also cellular debris and proteins from Sf9 culture, which can be toxic in vitro (personal observation) and may induce inflammation or an immune response when used in vivo. To avoid this, especially when using highly concentrated virus stocks, infectious baculovirus needs to be purified and separated from contaminating particles.
Several methods have been reported for the purification and concentration of baculovirus vectors10,11,12. Of the available approaches, heparin affinity chromatography allows for a single-step high level of purification with the low concentration of contaminating proteins12. The method is based on the identification of heparan sulfate as the receptor for baculovirus13,14. After loading Sf9 cell supernatant onto the column and binding of baculovirus, the column can be washed with physiologic (isotonic) buffer to remove unbound or loosely bound contaminating particles. Since the binding to heparin is reversible, baculovirus particles can be eluted with a high salt buffer, which is diluted immediately to physiologic (isotonic) salt concentration to prevent inactivation by osmotic shock12. Moreover, the production of baculovirus, as well as capture on and elution from the chromatography column, can be performed using a closed-system process which is compatible with current good manufacturing practices (cGMP).
Here, we provide a detailed protocol for the manufacture, purification, and concentration of infectious baculovirus using affinity chromatography and centrifugation. Briefly, we produce baculovirus by transfection of Sf9 cells with a baculovirus plasmid DNA in adherent culture and further expand the infectious baculovirus in serum-free suspension culture. We purify baculovirus using heparin affinity chromatography and use ultracentrifugation as the final step to highly concentrate the vector for gene therapy application.

<table><tbody><tr><td>Akta Avant 150</td><td>GE Healthcare</td><td>28976337</td><td>Chromatography system</td></tr><tr><td>POROS Heparin 50 &amp;micro;m Column</td><td>ThermoFisher Scientific</td><td>4333414</td><td>Heparin column</td></tr><tr><td>Ultracentrifuge</td><td>Beckman-Coulter</td><td>Non-catalog item</td><td>Concentrates virus at high-speed</td></tr><tr><td>Polyallomer Ultracentrifuge tube</td><td>Beckman-Coulter</td><td>326823</td><td>Concentrates virus at high-speed</td></tr><tr><td>MaxQ 8000 orbital shaker incubator&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>Non-catalog item</td><td>Shaker for suspension culture</td></tr><tr><td>250 mL Erlenmeyer flasks</td><td>ThermoFisher Scientific</td><td>238071</td><td>Flask for suspension culture</td></tr><tr><td>1 L Erlenmeyer flasks</td><td>ThermoFisher Scientific</td><td>238072</td><td>Flask for suspension culture</td></tr><tr><td>Microscope (Olympus CKX41)</td><td>Olympus</td><td>Non-catalog item</td><td>Cell monitoring and counting&amp;nbsp;</td></tr><tr><td>Table top centrifuge</td><td>ThermoFisher Scientific</td><td>75253839/433607</td><td>For clarification of Baculovirus supernatant</td></tr><tr><td>50 ml Conical tube</td><td>ThermoFisher Scientific</td><td>14-959-49A</td><td>For collection of Baculovirus supernatant</td></tr><tr><td>6-well plate</td><td>ThermoFisher Scientific</td><td>07-200-80</td><td>Tissue culture treated plate</td></tr><tr><td>10-cm plate</td><td>ThermoFisher Scientific</td><td>08-772E</td><td>Tissue culture treated plate</td></tr><tr><td>Stericup-HV, 0.45&amp;nbsp;&amp;micro;m, PVDF</td><td>EMD-Millipore</td><td>SCHVU05RE</td><td>Filtration unit</td></tr><tr><td>Kanamycin</td><td>ThermoFisher Scientific</td><td>15160-054</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma-Aldrich</td><td>T7660</td><td></td></tr><tr><td>Gentamycin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>Bac-to-Bac Vector Kit</td><td>ThermoFisher Scientific</td><td>10360-014</td><td>Baculovirus expression system</td></tr><tr><td>DH10B-T1R&amp;nbsp; Competent cell</td><td>ThermoFisher Scientific</td><td>12331-013</td><td>Competent cell for bacmid</td></tr><tr><td>TE buffer</td><td>In-house</td><td>Non-catalog item</td><td>10 mM Tris-HCl, 1 mM EDTA, pH 8.0.</td></tr><tr><td>Plasmid Maxiprep kit</td><td>ThermoFisher Scientific</td><td>K2100-06</td><td>For bacmid purification</td></tr><tr><td>Sf9 Cells</td><td>ThermoFisher Scientific</td><td>11496-015</td><td>Insect cells</td></tr><tr><td>Grace&amp;rsquo;s Insect Cell Culture Medium</td><td>ThermoFisher Scientific</td><td>11605-094</td><td>Transfection medium</td></tr><tr><td>PBS</td><td>ThermoFisher Scientific</td><td>20012227</td><td>Washing cells, diluting samples</td></tr><tr><td>HyClone SFX-Insect cell media</td><td>GE Healthcare</td><td>SH30278.02</td><td>Serum-free insect cell growth medium</td></tr><tr><td>Benzonase Nuclease</td><td>Sigma-Aldrich</td><td>E1014</td><td>Enzyme to degrade DNA and RNA</td></tr><tr><td>Baculovirus plasmid (bacmid) DNA</td><td>In-house</td><td>Non-catalog item</td><td>Bacmid for Baculovirus Production</td></tr><tr><td>Cellfectin II&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>10362</td><td>Transfection reagent for insect cells</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4737</td><td>Stabilizes Baculovirus</td></tr><tr><td>Cryovial</td><td>Thomas Scientific</td><td>1222C24</td><td>For storage of Baculovirus</td></tr><tr><td>HT1080 cell line</td><td>ATCC</td><td>CCL-121</td><td>Fibroblast cell line</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td>Growth media for cell lines</td></tr><tr><td>Wash buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride</td></tr><tr><td>Elution buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride</td></tr><tr><td>Column cleaning buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride</td></tr><tr><td>Sterile water</td><td>In-house</td><td>Non-catalog item</td><td>For Akta Avant cleaning</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>1.09137</td><td>For Akta Avant cleaning</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7073</td><td>For Akta Avant cleaning</td></tr></tbody></table>

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.

The protocol presented is in a flow diagram (Figure 1). Steps include the transient transfection of Sf9 cells with bacmid DNA to produce baculovirus in adherent culture in a plate, the subsequent amplification in serum-free suspension culture, nuclease treatment and clarification by centrifugation and filtration, and the purification using the heparin affinity chromatography followed by concentration with ultracentrifugation.
After thawing, Sf9 cells need at least two weeks to recover and entering into the exponential phase of growth. Actively growing Sf9 cells are transfected with bacmid DNA in adherent culture. Two days after transfection, expression of baculovirus envelope glycoproteins, gp64 is detected and subsequently, baculovirus production is initiated11. Since baculovirus is replication competent, the number of infected cells gradually increases due to a progressive infection of cells with the newly produced baculovirus that is secreted into the growth medium. Baculovirus containing cell supernatants are harvested 5 days after transfection when the entire cell population has become infected. This initial virus supernatant is designated as passage 1 (P1) stock. The P1 stock is used for the subsequent infection of a newly seeded adherent culture of Sf9 cells in a larger plate. The entire cell population shows sign of infection in 5 days which is called cytopathic effect (Figure 2). The supernatant from the second adherent cell culture is harvested and is designated as P2 stock. Baculovirus supernatant is further amplified in Sf9 suspension culture through ongoing infection of freshly added Sf9 cells in a shaker flask with serum-free insect cell culture medium. At the end of each infection cycle, most of the cells show cytopathic effect with an increased cellular diameter and dead or lysed cells, which are signs for completion of baculovirus infection. Baculovirus stock is sequentially amplified until the desired volume is obtained (P3, P4, &#8230;.). The supernatant is separated from the Sf9 cells by low-speed centrifugation, treated with a nuclease to reduce viscosity, filtered through a 0.45 &#956;m membrane, before being used in the subsequent purification and concentration steps.
To purify baculovirus, supernatant from infected Sf9 cells is loaded onto a heparin column. The heparin column gradually becomes saturated with strongly bound baculovirus and loosely bound protein contaminants. The heparin column is rinsed with wash buffer to remove contaminants until the ultraviolet (UV) absorbance curve (280 nm) returns to baseline and becomes stable, indicating the removal of unbound and loosely bound materials from the column. Baculovirus particles on the other hand strongly bind to the heparin column. Therefore, no significant amount of virus is detected in the column wash buffer. Heparin-bound baculovirus particles are eluted with 1.5 M of sodium chloride at pH 8.0 and diluted 10-fold with buffer to attain isotonicity and prevent virus inactivation. A sharp peak of protein is observed during elution which corresponds to the baculovirus fraction (Figure 3). The optimized conditions for sample loading, washing, and elution used in this chromatography protocol yield more than 50% recovery of purified infectious baculovirus particles. Diluted supernatants are further concentrated up to 500-fold with ultracentrifugation and formulated in PBS containing 0.5 % BSA, which yields a net 25% recovery11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57019/57019fig1.jpg" /> 
Figure 1. Schematic diagram for the production and purification of baculovirus. Sf9 cells are seeded in cell culture-treated plate transfected with bacmid DNA. Baculovirus supernatant is harvested and used to infect new Sf9 cells in adherent culture. Infected cells are subsequently propagated in serum-free suspension culture. Baculovirus supernatant is treated with a nuclease, centrifuged, and filtered. Baculovirus is purified by heparin affinity column chromatography, diluted in buffer, and concentrated aseptically by ultracentrifugation. Finally, baculovirus supernatant is stored at -80 &#176;C. (The figure has been adapted from Nasimuzzaman et al., 2016, Mol Ther Methods Clin Dev. 2016; 3: 16071 with permission.) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57019/57019fig1large.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/57019/57019fig2.jpg" /> 
Figure 2. Cytopathic effects of baculovirus infected Sf9 cells. Baculovirus supernatant was used for infecting the Sf9 cells in a tissue-culture treated plate. Five days after infection, cells were visualized with an inverted phase microscope. A) Uninfected Sf9 cells as a control. B) Baculovirus infected Sf9 cells showing cytopathic effect. Cells are shown at 200X magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57019/57019fig2large.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/57019/57019fig3.jpg" /> 
Figure 3. The number of infectious baculovirus in flow-through during heparin affinity chromatography. Baculovirus titers were estimated by infection of HT1080 cells. Samples tested were including the column run-through, wash, and eluate. Samples were diluted as needed. The line (dark diamonds) shows the total infectious units (IU) of baculovirus in each fraction. The fraction size of column flow-through sample and wash buffer were 40 mL in each tube, and eluate was 10 mL in each tube. (The figure has been adapted from Nasimuzzaman et al., 2016, Mol Ther Methods Clin Dev. 2016; 3: 16071 with permission.) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57019/57019fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Production and Purification of Baculovirus 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 ...

production-purification-baculovirus-for-gene-therapy

Nanyang Technological University

Research

JoVE Journal

Genetics

14.6K Views.  Cincinnati Children's Hospital Medical Center. In dit protocol is baculovirus geproduceerd door voorbijgaande transfectie van baculovirus plasmide in Sf9 cellen en versterkt in een serumvrij schorsing cultuur. De bovendrijvende substantie is gezuiverd door de chromatografie van de affiniteit van heparine en verder geconcentreerd door ultracentrifugatie. Dit protocol is handig voor de productie en zuivering van baculovirus voor gen-therapie-toepassing.

Video: Production and Purification of Baculovirus for Gene Therapy Application

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 ...

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- ...

Biochemistry

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 ...

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.

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. ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

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 ...

Biology

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 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

Production and Purification of Baculovirus for Gene Therapy Application

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