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 generate insect expression plasmids harboring chimeric proteins, with the fluorescent proteins fused in-frame to the carboxyl terminus of the genes of interest (in this case, to two Bemisia tabaci aquaporin proteins) or to subcellular marker proteins (Figure 1).
<ol>
	<li>Use OE-PCR, which consists of two independent rounds of amplification, to generate: (1) sequences encoding the genes of interest (B. tabaci aquaporin 1: BtDrip1; B. tabaci aquaporin 2: BtDrip2_v1) fused in-frame with a green fluorescent protein variant called EGFP or (2) the subcellular markers (Drosophila melanogaster sex peptide receptor: DmSPR; Homo sapiens phospholipase A2: HsPLA2) with the mCherry coding sequence. Directly ligate the final OE-PCR products into the pIB/V5-His-TA plasmid.
	<ol>
		<li>For the first round of OE-PCR (indicated by A-D and A&#39;-D&#39; in Figure 1), use a gene-specific sense primer (shown in bold in Table 1) and a chimeric antisense primer (italicized in Table 1) corresponding to the final 15 bp (without the stop codon) of the gene of interest/subcellular marker and to 15 bp of the 5&#39;-end of the desired fluorescent protein (EGFP or mCherry) to generate a product with a 3&#39; overhang. 
		Note: See Table 2 for PCR conditions.</li>
		<li>In a separate tube, use a gene-specific fluorescent protein antisense primer (shown with an underline in Table 1) and a chimeric sense primer that contains 15 bp from the 3&#39;-end of the gene of interest/subcellular marker and 15 bp from the 5&#39;-end of the desired fluorescent protein to generate a product with a 5&#39; overhang. 
		Note: See Table 2 for the PCR conditions. The PCR template can either be a sequence-validated PCR product or, more preferably, plasmid DNA harboring the desired sequence. The study described here uses sequence-validated plasmids.</li>
		<li>Separate the PCR products with 1% agarose gel electrophoresis (using standard molecular-grade agarose).</li>
		<li>Excise and purify the products of the expected sizes (see Table 2) using a commercially available DNA gel extraction kit (see the table of materials for the specific kit used in this protocol).</li>
		<li>Use the gel-purified PCR products from step 1.1.4 to generate the final overlap extension products (indicated by A&#39;&#39;-D&#39;&#39; in Figure 1). Use a gene-specific sense primer for the gene of interest/subcellular marker and the corresponding gene-specific antisense primer for the fluorescent protein to generate the desired chimeric sequence. 
		Note: See Table 2 for the PCR conditions. It may be necessary to empirically determine the optimal amounts of first-round overlap extension products to add to the reaction mixture to yield the final, full-length chimeric PCR product.</li>
		<li>Separate the second-round OE-PCR products by 1% agarose gel electrophoresis. Excise and purify the products using a commercially available DNA gel extraction kit.</li>
		<li>Incubate gel-purified second-round OE-PCR products with 1 unit of Taq DNA polymerase master mix for 10 min at 72 &#176;C to generate the 3&#39; adenylated (i.e. A-tailed)&#160;products needed for TA cloning.</li>
		<li>Ligate the resulting adenylated products into the pIB expression vector and use standard molecular cloning techniques to transform chemically competent (heat shock) or electrocompetent (electroporation) Escherichia coli cells. Plate overnight on medium containing the appropriate selection marker (i.e., ampicillin or carbenicillin).</li>
		<li>Perform colony PCR12 using a vector-specific primer and an insert-specific primer to identify insert-positive colonies that have been transformed with expression plasmids harboring an insert in the 5&#39;-3&#39; orientation. Prepare overnight cultures of colonies generating PCR products of the expected sizes.</li>
		<li>Isolate plasmid DNA using a commercially available DNA purification kit according to the manufacturer&#39;s instructions (see the Materials Table for the specific kit used in this protocol). Validate the sequence integrity of each insert by direct DNA sequencing.</li>
	</ol>
	</li>
</ol>
2. Insect Cell Culture Maintenance
Note: To maintain sterile conditions, conduct all cell manipulations requiring the opening of the tissue culture flask within a laminar flow hood. Turn on the laminar flow hood UV germicidal lamp at least 1 h prior to cell manipulations. Wear nitrile gloves and decontaminate the surface of the bench, pipettes, utensils, tubes, and flasks with 70% ethanol before their use. Familiarity with basic cell culture techniques is recommended13.
<ol>
	<li>Use Tni cells (an established cell culture line derived from Trichoplusia ni ovarian tissue) that are adapted to serum-free medium and maintain them as an adherent monolayer culture in serum-free insect medium at 28 &#176;C in T25 tissue culture flasks.</li>
	<li>Maintain Sf9 cells (an established cell culture line derived from Spodoptera frugiperda ovarian tissue) similarly but using TNM-FH insect culture medium supplemented with 10% Fetal Bovine Serum (FBS).</li>
	<li>Seed the initial culture from frozen Sf9 or Tni cells by removing stock vials from -80 &#176;C and allowing them to thaw in a 37 &#176;C water bath. Decontaminate the vials with 70% ethanol after thawing and place them on ice.</li>
	<li>Add 4 mL of insect cell medium to a new T25 flask and transfer 1 mL of the thawed insect cell suspension. Place the flask in a 28 &#176;C, non-humidified incubator and allow the cells to attach for 30-45 min.</li>
	<li>Replace the seeding medium with 5 mL of the appropriate medium and transfer the flasks to a 28 &#176;C non-humidified incubator. Monitor cell confluence daily. Passage the cells when they reach 90% confluency. 
	Note: Complete coverage of T25 flask corresponds to ~5 x 106 cells.</li>
	<li>Insect cell passage.
	<ol>
		<li>To passage the cells, first remove the exhausted medium from the flask containing confluent cells using a sterile 5 mL serological pipette. Tilt the flask so that the medium flows to one corner, away from the cell monolayer. Carefully remove the medium using a pipette without disturbing the cells.</li>
		<li>Dislodge the Tni cells by gently rinsing the T25 flasks containing the confluent monolayer with 4 mL of serum-free insect medium using new, sterile, 5 mL serological pipette. Move the pipette tip across the flask and slowly irrigate to remove cells loosely attached to the flask bottom.
		<ol>
			<li>Check for the adequate detachment of cells by removing all media, turning the flask over, and observing that the bottom of the flask is clear.</li>
		</ol>
		</li>
		<li>For Sf9 cells, which adhere more tightly, add 4 mL of fresh TNM-FH medium and use a cell scraper to dislodge the attached cells. Use a 5 mL serological pipette to gently mix and reduce cell clumping.</li>
		<li>Use an automated cell counter to estimate the number of viable insect cells per volume of medium. Transfer 0.1 mL of cell/medium mixture to a 1.5-mL microfuge tube. In a separate 0.5 mL microfuge tube, add 10 &#181;L of cell/medium mixture to 10 &#181;L of trypan blue.</li>
		<li>Remove a cell counter chamber slide from its packaging and add 10 &#181;L of the cell/medium/trypan blue mixture to each side of the counting slide. Insert the slide into the cell counter and determine the cell density and viability.</li>
		<li>Using the cell density, calculate and add the proper volume of insect cell medium (up to 5 mL) to new, sterile T25 flasks.</li>
		<li>Transfer approximately 1-1.5 x 106 cells to T25 flasks with fresh media; label the flasks with the cell line, date, medium used, number of cells added, and passage number (Pn + 1 generation, where Pn is the passage number for the previous generation of cells); and place the flasks in a 28 &#176;C incubator for up to 72 h. 
		Note: Insect cells may be continuously propagated, although cells may be less receptive to transfection and/or heterologous protein expression after 30 passages. Treat all discarded cells/media with 10% bleach solution and autoclave disposable plastic ware before disposal.</li>
	</ol>
	</li>
</ol>
3. Insect Cell Transfection
<ol>
	<li>Seed a T25 flask with up to 1 x 106 Tni or Sf9 cells in an appropriate insect cell medium (serum-free insect medium for Tni and TNM-FH for Sf9) and grow to confluency for 72 h at 28 &#176;C.</li>
	<li>Remove and discard the old medium and dislodge the cells with 4 mL of fresh serum-free insect medium (see steps 2.6.2 and 2.6.3, above).</li>
	<li>Estimate the cell density using an automated cell counter (see step 2.6.4, above).</li>
	<li>Add approximately 7 x 105 cells to individual 35 mm glass-bottom dishes and allow the cells to attach for 20-25 min at 28 &#176;C.</li>
	<li>For each transfection, add 2 &#181;g of plasmid DNA (either from one plasmid for single transfections or 2 &#181;g from each of the two plasmids&#160;for double transfections) to 0.1 mL of serum-free insect medium (without FBS for both Sf9 and Tni transfections) in a sterile 1.5 mL microfuge tube.</li>
	<li>In a separate tube, mix 8 &#181;L of transfection reagent with 0.1 mL of serum-free insect medium and then transfer that solution to the tube containing the plasmid DNA of interest. Lightly vortex and incubate at RT for 20 - 30 min.</li>
	<li>Dilute the plasmid-transfection mixture from steps 3.5 and 3.6 with 0.8 mL of serum-free insect medium so that the total volume equals 1 mL.</li>
	<li>Carefully remove the media from the glass dishes containing attached cells. Overlay the attached cells with the diluted plasmid-transfection medium.</li>
	<li>Incubate the cells at 28 &#176;C for 5 h. 
	Note: The conditions for transfection require empirical optimization for maximal transfection efficiency (e.g.,&#160;overnight&#160;transfection rather than 5 h, the amount of plasmid DNA used, the chemistry of the transfection reagent used, etc.).</li>
	<li>Remove and discard the transfection medium and gently wash the cells with 1 mL of serum-free insect medium, being careful not to dislodge cells.</li>
	<li>Add 2 mL of fresh insect cell medium (serum-free insect medium for Tni and TNM-FH for Sf9) and incubate at 28 &#176;C for 48-72 h. 
	Note: Again, conditions require empirical optimization for maximal heterologous protein expression.</li>
</ol>
4. Confocal Fluorescence Microscopy
<ol>
	<li>At 48-72 h post-transfection, wash the cells once with 1 mL of IPL-41 insect medium and then cover with 2 mL of IPL-41 for imaging. 
	Note: This wash step reduces the background auto-fluorescence observed with insect cell medium used in normal cell maintenance.</li>
	<li>Add 4 drops of Hoechst live-cell staining reagent (see the table of materials for the specific nuclear stain used in this protocol) to the medium and incubate at 28 &#176;C for 20-25 min. 
	Note: Additional nuclear stains may be substituted, although the dye should have a fluorescence profile unique from EGFP and mCherry.</li>
	<li>Place a 35 mm dish into the self-enclosed laser scanning confocal microscope (see the table of materials for the specific instrument used in this protocol).</li>
	<li>Adjust the microscope for Hoechst, EGFP, and mCherry observation conditions: Hoechst excitation/emission -&#160;359/461 nm; EGFP excitation/emission -&#160;489/510 nm; mCherry excitation/emission -&#160;580/610 nm.</li>
	<li>Perform an initial scan using a 10x objective to confirm fluorescent expression and then switch to scanning mode using a 60X phase contrast water-immersion objective.</li>
	<li>Adjust the laser power (5-7%), detector sensitivity (47-49%), 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. 
	Note: Microscope parameters require empirical adjustment for optimal image collection and may be specific to the instrument in use.</li>
	<li>Export the raw data as TIFF image files and modify (crop and overlay) for figure generation.</li>
</ol>

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The authors declare that they have no competing financial interests.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

transient-expression-cellular-localization-recombinant-proteins

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.6K Views.  Agricultural Research Service, U.S. Department of Agriculture. 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.

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

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

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

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

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

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 ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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

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

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

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

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

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

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Chapters in this video

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

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

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

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

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension