Name: transient expression and cellular localization of recombinant proteins in cultured insect cells
Uploaded: 2017-04-20T15:00:00
Description: Watch this Scientific Journal Video about Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells at JoVE.com

We thank Lynn Forlow-Jech and Dannialle LeRoy for technical assistance. This work was supported by base CRIS funding to USDA ARS, National Program 304 - Crop Protection and Quarantine [Project #2020-22620-022-00D] to J.A.F. and J.J.H. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture. USDA is an equal opportunity provider and employer.

1. OE-PCR for the Construction of Expression Plasmids
Note: See Table 1 for all primers used in OE-PCR. The use of a high-fidelity DNA polymerase is recommended for all amplifications. However, because these enzymes frequently do not leave a 3&#39; A, it is necessary to perform a brief, non-amplifying incubation with a Taq DNA polymerase to &#34;A-tail&#34; the PCR products prior to cloning them into a TA insect cell expression vector. This protocol demonstrates a method to gene...

<ol>
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Virol. 96 (1), 6-23 (2015).</li><li>Contreras-G&#242;mez, A., 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=Protein+production+using+the+baculovirus-insect+cell+expression+system.">Protein production using the baculovirus-insect cell expression system.</a> Biotechnol. Progr. 30 (1), 1-18 (2014).</li><li>Hunt, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gene+to+protein:+a+review+of+new+and+enabling+technologies+for+multi-parallel+protein+expression.">From gene to protein: a review of new and enabling technologies for multi-parallel protein expression.</a> Protein Expres. 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Biotechnol. 216, 67-75 (2015).</li><li>Chen, H., 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=Rapid+screening+of+membrane+protein+expression+in+transiently+transfected+insect+cells.">Rapid screening of membrane protein expression in transiently transfected insect cells.</a> Protein Expres. Purif. 88 (1), 134-142 (2013).</li><li>Shen, X., 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=Virus-free+transient+protein+production+in+Sf9+cells.">Virus-free transient protein production in Sf9 cells.</a> J. Biotechnol. 171, 61-70 (2014).</li><li>Loomis, K. 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Heterologous protein expression systems are important tools for the production of recombinant proteins used in numerous downstream applications4. Choosing from the diverse expression systems available depends on the end goal for the protein of interest. Several insect cell expression systems are available that offer flexible alternatives to prokaryotic and eukaryotic cell expression systems5,6. Insect systems requiring baculovirus infectio...

The production of recombinant proteins using insect cell expression systems offers numerous benefits for the study of eukaryotic proteins. Namely, insect cells possess similar post-translational modifications, processing, and sorting mechanisms as those present in mammalian cells, which is advantageous for producing properly folded proteins1,2,3. Insect cell systems also typically require fewer resources and less time and effort for maintenance than mammalian cell lines4,5. The baculovirus expression system is one such insect cell-based system that is now widely used in many disciplines, including the production of recombinant proteins for protein characterization and therapeutics, the immunogenic presentation of foreign peptides and viral proteins for vaccine production, the synthesis of multi-protein complexes, the production of glycosylated proteins, etc.1,2,4,6. There are, however, situations in which baculovirus expression may not be applicable3,7, and the use of nonlytic and transient insect expression systems may be more appropriate. Specifically, transient insect cell expression offers the possibility for the rapid synthesis of recombinant protein, requires less development and maintenance, does not involve viral-imposed cell lysis, and provides a means to better study cellular trafficking during protein synthesis7,8,9,10.
This protocol describes the rapid generation of expression vectors using two-step overlap extension PCR (OE-PCR) 11 and the standard cloning of plasmid DNA in Escherichia coli. Plasmids are used to double-transfect commercially available cultured insect cells and to produce representative proteins. The protocol describes the production and use of two different fluorescently-labeled subcellular marker proteins and demonstrates colocalization with two aquaporin proteins from the insect Bemisia tabaci. The following protocol provides the basic methodology for OE-PCR, insect cell maintenance and transfection, and fluorescence microscopy for the cellular localization of target proteins.

<table border="0" cellpadding="0" cellspacing="0" width="1353">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">KOD DNA Polymerase</td>
			<td style="width:164px;">EMD Millipore</td>
			<td style="width:119px;">71085-3</td>
			<td style="width:645px;">High-fidelity DNA polymerase used for PCR amplification of overlap extension PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">ExTaq DNA Polymerase</td>
			<td style="width:164px;">TaKaRa-Clontech</td>
			<td style="width:119px;">RR001B</td>
			<td>DNA polymerase used for A-tailing of PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">EconoTaq PLUS GREEN 2x DNA Polymerase Master Mix</td>
			<td style="width:164px;">Lucigen</td>
			<td style="width:119px;">30033-1</td>
			<td>DNA polymerase used for bacterial colony PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Biometra TProfessional Gradient Thermocycler</td>
			<td style="width:164px;">Biometra/LABRepCo</td>
			<td style="width:119px;">070-851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Agarose LE</td>
			<td style="width:164px;">Benchmark Scientific</td>
			<td style="width:119px;">A1705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">SYBR Safe DNA Gel Stain</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Montage DNA Gel Extraction Kit</td>
			<td style="width:164px;">EMD Millipore</td>
			<td style="width:119px;">LSKGEL050</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">pIB/V5-His TOPO TA Expression Kit</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">K89020</td>
			<td style="width:645px;">Contains components needed to clone overlap extension PCR products, including linearized and topoisomerase I-activated pIB/V5-His-TOPO vector, One Shot TOP10 chemically competent E. coli, and salt solution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">QIAprep Spin MiniPrep Kit</td>
			<td>Qiagen</td>
			<td style="width:119px;">27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">QIAcube Robotic Workstation</td>
			<td>Qiagen</td>
			<td style="width:119px;">9001292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purifier Vertical Clean Bench</td>
			<td>Labconco</td>
			<td>3970401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tni cultured insect cell Line</td>
			<td>Allele Biotech</td>
			<td>ABP-CEL-10005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sf9 cultured insect cell Line</td>
			<td>Allele Biotech</td>
			<td>ABP-CEL-10002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-Free Insect Culture Medium</td>
			<td>Allele Biotech</td>
			<td>ABP-MED-10002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TNM-FH Insect Culture Medium</td>
			<td>Allele Biotech</td>
			<td>ABP-MED-10001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPL-41 Insect Medium</td>
			<td>ThermoFisher</td>
			<td>11405081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellfectin II Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>10362100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16 cm Disposable Cell Scrapers</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td>Cell scrapers with two-position blade</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">25 cm2 (T25) Tissue Culture Flasks with Vent Filter Caps</td>
			<td>Life Science Products</td>
			<td>CT-229331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer Pipets</td>
			<td>Fisher</td>
			<td>1371120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, 50 mL Bio-Reaction Tubes</td>
			<td>Life Science Products</td>
			<td>CT-229475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PipetteBoy</td>
			<td>VWR</td>
			<td>14222-180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL Serological Pipettes</td>
			<td>Sarstedt</td>
			<td>86.1253.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL Flat-Cap PCR Tubes</td>
			<td>Fisher</td>
			<td>14230200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene Biohazard Autoclave Bags</td>
			<td>Fisher</td>
			<td>01828C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm #1.5 Glass Bottom Dishes</td>
			<td>Matsunami Glass</td>
			<td>D35-14-1.5-U</td>
			<td>35 mm dish diameter, 14 mm glass diameter, 1.5 mm glass thickness, uncoated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator, Model 1510E</td>
			<td>VWR</td>
			<td>35823-961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess II FL Cell Counter</td>
			<td>ThermoFisher</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess Cell Counting Chamber Slides with 0.4% Trypan Blue Reagent</td>
			<td>ThermoFisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoview FV10i-LIV Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV10i-LIV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HsPLA2/pCS6 plasmid DNA</td>
			<td>transOMIC Technologies</td>
			<td>TCH1303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmCherry Vector</td>
			<td>Clontech</td>
			<td>632522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td>
			<td style="width:164px;">ThermoFisher</td>
			<td style="width:119px;">R37605</td>
			<td></td>
		</tr>
	</tbody>
</table>

transient expression and cellular localization of recombinant proteins in cultured insect cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

OE-PCR
OE-PCR allows for the synthesis of chimeric DNA products that, once inserted into an expression vector, allow for the production of recombinant chimeric proteins corresponding to any test gene of interest and fluorescent marker protein. Figure 1 represents a general scheme for the production of pIB expression vectors containing B. tabaci aquaporin coding sequences (BtDrip1 and BtDrip2_v1) in-frame w...

Watch this Scientific Journal Video about Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells at JoVE.com

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

The overall goal of this procedure is to express fluorescently tagged proteins of interest within commercially available insect cells for the enhanced elucidation of protein function and or cellular trafficking of proteins. This method can help answer key cell biology and biochemistry based questions regarding protein functionality, protein interactions, and or subcellular protein localization. The main advantage of this technique is that constructs can be rapidly generated and expressed proteins functionally assessed in live cells without deleterious effects associated with baculovirus expression, thus improving throughput.Though this method can provide insight in the study of insect proteins, it can also be applied to any system for which the study of protein function or cellular localization is desired. Demonstrating the procedure for insect cell culture and transfection will be Danni LeRoy, a technician from my laboratory. To begin this procedure, remove stock vials of frozen SF9 and Tni cells from the minus 80 degrees celsius freezer and allow them to thaw in a 37 degree celsius water bath.After thawing, decontaminate the vials with 70%ethanol and place them on ice. All cell manipulations requiring the opening of vials and tissue culture flasks must be performed within a laminar flow hood to maintain sterile conditions. Add four milliliters of serum-free insect medium to a new T25 flask and four milliliters of TNM-FH medium to another T25 flask.Transfer one milliliter of thawed Tni cells to the flask with serum-free insect medium and one milliliter of thawed SF9 cells to the flask with TNM-FH medium. Place the flasks in a 28 degree celsius non-humidified incubator and allow the cells to attach for 30 to 45 minutes. Replace the seeding medium with five milliliters of the appropriate medium.And return the flasks to the 28 degree celsius non-humidified incubator. Monitor cell confluence daily. Passage the cells when they reach 90%confluency as shown by these representative images.Tilt the flask containing the confluent cells so that the medium flows toward one corner away from the cell monolayer and use a sterile five milliliter serological pipette to carefully remove the medium without disturbing the cells. For Tni cells, use a new sterile five milliliter serological pipette to gently add four milliliters of serum-free insect medium onto the confluent monolayer. Move the pipette tip across the flask and slowly irrigate to remove cells loosely attached to the flask bottom.To check for the adequate detachment of cells, remove all media, turn the flask over and observe that the bottom of the flask is clear. For SF9 cells, which adhere more tightly, add four milliliters of fresh TNM-FH medium and use a cell scraper to dislodge the attached cells. Use a five milliliter serological pipette to gently mix and reduce cell clumping.After detaching the cells, transfer approximately 0.1 milliliters of each cell and medium mixture to a 1.5 milliliter micro-fuge tube. In a separate 0.5 milliliter micro-fuge tube add 10 microliters of each cell and medium mixture to 10 microliters of trypan blue. Remove the cell counter chamber slide from its packaging and add 10 microliters of the cell medium trypan blue mixture to each side of the counting slide.Insert the slide into an automated cell counter and determine the cell density and viability. Transfer approximately one to 1.5 times 10 to the 6th cells to T25 flasks with fresh media. Label the flasks with the cell line, date, medium used, number of cells added, and passage number.Place the flasks in a 28 degree celsius incubator for up to 72 hours. The procedure for insect cell transfection must also be performed within a laminar flow hood. Seed a T25 flask with up to one times 10 to the 6th Tni or SF9 cells in an appropriate insect cell medium.Tni cells are used in this demonstration. Grow the cells to confluency for 72 hours at 18 degrees celsius. 72 hours later, remove and discard the old medium and dislodge the Tni cells with four milliliters of fresh serum-free insect medium as shown earlier.The most difficult aspect of this procedure is obtaining the appropriate density of cells attached to the glass bottom dishes during transfection. No more than seven times 10 to the 5th cell should be added to each dish. After using an automated cell counter to estimate the cell density, thoroughly but gently mix the cell suspension by inverting the tube and add approximately seven times 10 to the 5th cells to individual 35 millimeter glass bottom dishes.Allow the cells to attach for 20 to 25 minutes at 28 degrees celsius. For each transfection, add two micrograms of plasmid DNA to 0.1 milliliters of serum-free insect medium without FBS in a sterile 1.5 milliliter micro-fuge tube. In a separate tube, mix eight microliters of transfection reagent with 0.1 milliliters of serum-free insect medium.Then transfer the solution to the tube containing the plasmid DNA of interest. Lightly vortex and incubate at room temperature for 20 to 30 minutes. Next, dilute the plasmid transfection mixture with 0.8 milliliters of serum-free insect medium, bringing the total volume up to one milliliter.Carefully remove the media from the glass dish containing attached cells. Overlay the attached cells with the diluted plasmid transfection medium. Incubate the cells at 28 degrees celsius for five hours.After five hours, remove and discard the transfection medium and gently wash the cells with one milliliter of serum-free insect medium, being careful not to dislodge the cells. Add two milliliters of fresh serum-free insect cell medium and incubate at 28 degrees celsius for 48 to 72 hours. 48 to 72 hours after transfection of the insect cells, wash the cells once with one milliliter of IPL-41 insect medium and then cover with two milliliters of IPL-41 for imaging.Add four drops of Hoechst live cell staining reagent to the medium and incubate at 28 degrees celsius for 20 to 25 minutes. Place the 35 millimeter dish into the self enclosed laser scanning confocal microscope. Although many glass dishes are commercially available, it's important that they fit the microscope stage and it may be necessary to empirically determine which glassware is most compatible with cell attachment.Adjust the microscope for Hoechst, EGFP and mCherry observation conditions. 359 nanometers and 461 nanometers for Hoechst excitation and emission. 489 nanometers and 510 nanometers for EGFP excitation and emission.And 580 nanometers and 610 nanometers for mCherry excitation and emission. Using a 10X objective, perform an initial scan to confirm fluorescent expression. After that, switch to scanning mode using a 60X phase contrast water immersion objective.Adjust the laser power, detector sensitivity, scanning speed, Z-axis depth, and digital zoom to optimize the image contrast and resolution. Image the cells at 1.5X digital zoom to give a total of 90X amplification. Save and export the raw data as TIFF image files for later analysis.Tni cells were transfected with the indicated plasmids and the expression of recombinant proteins was visualized using confocal fluorescence microscopy. Successful transfection and expression of recombinant BtDrip1-EGFP is evident by the presence of green fluorescence on the cell's surface. Cells transfected with BtDrip2 version one EGFP show green fluorescence within indicating the intracellular expression of BtDrip2 version one.Likewise, cells transfected with PIB DmSPR-mCherry or PIB PLA2G15-mCherry show red fluorescence indicating the expression of the respective chimeras. In the merged images, the orange or yellow areas indicate expression of both EGFP and mCherry which suggests that the proteins are co-localized within the same subcellular structures. An overlay of cells double transfected with PIB BtDrip1-EGFP and PIB DmSPR-mCherry suggests co-localization of BtDrip1-EGFP and DmSPR-mCherry on the cell's surface.Cells double transfected with BtDrip2 version one EGFP and PIB DmSPR-mCherry show little co-localization of green and red fluorescence signals confirming the intracellular expression of BtDrip2 version one. In contrast the co-expression of PIB BtDrip2 version one EGFP and the PIB PLA2G15-mCherry lysosomal marker resulted in a significant overlap in the cytoplasmic green and red fluorescent signals. This strongly suggests that BtDrip2 is traffic to intercellular lysosomes.After watching this video, you should have a good understanding of how to express fluorescent protein chimeras within cultured insect cells. This system offers rapid vector construction in protein synthesis, avoids challenges of virus based expression systems, and provides a robust means to observe cellular trafficking proteins.

Research

JoVE Journal

Biology

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