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><tbody><tr><td>KOD DNA Polymerase</td><td>EMD Millipore</td><td>71085-3</td><td>High-fidelity DNA polymerase used for PCR amplification of overlap extension PCR products</td></tr><tr><td>ExTaq DNA Polymerase</td><td>TaKaRa-Clontech</td><td>RR001B</td><td>DNA polymerase used for A-tailing of PCR products</td></tr><tr><td>EconoTaq PLUS GREEN 2x DNA Polymerase Master Mix</td><td>Lucigen</td><td>30033-1</td><td>DNA polymerase used for bacterial colony PCR</td></tr><tr><td>Biometra TProfessional Gradient Thermocycler</td><td>Biometra/LABRepCo</td><td>070-851</td><td></td></tr><tr><td>Agarose LE</td><td>Benchmark Scientific</td><td>A1705</td><td></td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>ThermoFisher</td><td>S33102</td><td></td></tr><tr><td>Montage DNA Gel Extraction Kit</td><td>EMD Millipore</td><td>LSKGEL050</td><td></td></tr><tr><td>pIB/V5-His TOPO TA Expression Kit</td><td>ThermoFisher</td><td>K89020</td><td>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><td>QIAprep Spin MiniPrep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>QIAcube Robotic Workstation</td><td>Qiagen</td><td>9001292</td><td></td></tr><tr><td>Purifier Vertical Clean Bench</td><td>Labconco</td><td>3970401</td><td></td></tr><tr><td>Tni cultured insect cell Line</td><td>Allele Biotech</td><td>ABP-CEL-10005</td><td></td></tr><tr><td>Sf9 cultured insect cell Line</td><td>Allele Biotech</td><td>ABP-CEL-10002</td><td></td></tr><tr><td>Serum-Free Insect Culture Medium</td><td>Allele Biotech</td><td>ABP-MED-10002</td><td></td></tr><tr><td>TNM-FH Insect Culture Medium</td><td>Allele Biotech</td><td>ABP-MED-10001</td><td></td></tr><tr><td>IPL-41 Insect Medium</td><td>ThermoFisher</td><td>11405081</td><td></td></tr><tr><td>Cellfectin II Transfection Reagent</td><td>ThermoFisher</td><td>10362100</td><td></td></tr><tr><td>16 cm Disposable Cell Scrapers</td><td>Sarstedt</td><td>83.1832</td><td>Cell scrapers with two-position blade</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; (T25) Tissue Culture Flasks with Vent Filter Caps</td><td>Life Science Products</td><td>CT-229331</td><td></td></tr><tr><td>Transfer Pipets</td><td>Fisher</td><td>1371120</td><td></td></tr><tr><td>Sterile, 50 mL Bio-Reaction Tubes</td><td>Life Science Products</td><td>CT-229475</td><td></td></tr><tr><td>PipetteBoy</td><td>VWR</td><td>14222-180</td><td></td></tr><tr><td>5 mL Serological Pipettes</td><td>Sarstedt</td><td>86.1253.001</td><td></td></tr><tr><td>0.5 mL Flat-Cap PCR Tubes</td><td>Fisher</td><td>14230200</td><td></td></tr><tr><td>Polypropylene Biohazard Autoclave Bags</td><td>Fisher</td><td>01828C</td><td></td></tr><tr><td>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><td>Incubator, Model 1510E</td><td>VWR</td><td>35823-961</td><td></td></tr><tr><td>Countess II FL Cell Counter</td><td>ThermoFisher</td><td>AMQAF1000</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides with 0.4% Trypan Blue Reagent</td><td>ThermoFisher</td><td>C10228</td><td></td></tr><tr><td>Fluoview FV10i-LIV Laser Scanning Confocal Microscope</td><td>Olympus</td><td>FV10i-LIV</td><td></td></tr><tr><td>HsPLA2/pCS6 plasmid DNA</td><td>transOMIC Technologies</td><td>TCH1303</td><td></td></tr><tr><td>pmCherry Vector</td><td>Clontech</td><td>632522</td><td></td></tr><tr><td>NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td><td>ThermoFisher</td><td>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

Research

JoVE Journal

Biology

10.7K Views.  Agricultural Research Service, U.S. Department of Agriculture.  Nonlytic insectencel expressiesystemen onderbenut productie, cellulair transport / lokalisatie en recombinante eiwitten functionele analyse. We beschrijven werkwijzen voor expressievectoren en transiënte daaropvolgende eiwitexpressie genereren handel verkrijgbaar Lepidoptera cellijnen. De co-localisatie van Bemisia tabaci aquaporins met fluorescente subcellulaire markereiwitten wordt gepresenteerd. 

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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Genetics

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 ...

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 ...

Biochemistry

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

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

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

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

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

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 ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

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 ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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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