We thank Dr. Jian-Horng Leu of Institute of Marine Biology, National Taiwan Ocean University for providing 3 plasmid constructions. This research was supported by Grant 106-2311-B-197 -001 - from the Ministry of Science and Technology (MOST).

1. Preparations
<ol>
	<li>Insect cell culture
	<ol>
		<li>Prepare 50 mL of cell culture medium. To do so, add 500 &#181;L of antibiotics (Amphotericin B = 0.25 &#181;g/mL, Penicillin = 100 unit/mL, Streptomycin = 100 &#181;g/mL) and 5 mL of heat-inactivated fetal bovine serum in serum-free cell culture medium (without FBS or antibiotics). 
		NOTE: Heat the fetal bovine serum at 65 &#176;C for 30 min in a water bath before use.</li>
		<li>Maintain Spodoptera frugiperda (Lepidoptera: Noctuidae), sf9 insect cells. Detach ca. 80% of the cells from 25 cm2 cell culture flask by shaking the flask and check under light microscopy. Then, transfer 50% of the cell suspension to a new 25 cm2 cell culture flask and allow the cells to attach for 15 min at room temperature. Replace the medium with 5 mL fresh cell culture medium and grow in an incubator at 28 &#176;C. Passage cells every 2 to 3 days depending on the cell growth.</li>
	</ol>
	</li>
	<li>Preparation of plasmids for cell transfection
	<ol>
		<li>Insert the target DNA fragments (e.g., Lymantria xylina MNPV (LyxyMNPV) iap3 gene and its deletion constructs) into pDHsp/V5-His by PCR-based cloning method11. Check the growth of transformed colonies by colony PCR using PCR Master Mix (2X) and the pDhsp-F2/Op-IE2R primer set. Confirm the plasmid sequences by commercial sequencing service. 
		NOTE: Table 1 lists the primers used for PCR and the corresponding constructs.</li>
		<li>Culture single sequenced bacterial colonies, which contain the aforementioned plasmid constructions (step 1.2) in 200 mL LB medium containing selected antibiotics (50 &#181;g/mL), respectively.</li>
		<li>Extract the plasmids from the cultured E. coli using Midi Plasmid Kit according to manufacturer&#39;s instructions12.</li>
	</ol>
	</li>
	<li>Prepare plating medium by mixing 1.5 mL of cell culture medium and 8.5 mL of serum-free cell culture medium.</li>
	<li>Prepare Actinomycin D (ActD) cell culture medium by adding 1.5 &#181;L of ActD stock (1 mg/mL) into 10 mL of cell culture medium (final concentration = 150 ng/mL). Store at 4 &#176;C.</li>
</ol>
2. Protein transient expression
<ol>
	<li>Cell seeding
	<ol>
		<li>Harvest the sf9 cells by shaking culture flask, topple and fall the cell suspension to 50 mL tube, and transfer 10 &#181;L to hemocytometer by a P10 pipette. Count the cell number under a light microscope.</li>
		<li>Plate 3 &#215; 105 sf9 cells into each well in a 24-well plate for 15 min at room temperature. Replace the medium with 0.5 mL of plating medium.</li>
	</ol>
	</li>
	<li>Transfection of plasmids
	<ol>
		<li>Dilute cell transfection reagent: Dilute 8 &#181;L of cell transfection reagent in 100 &#181;L serum-free cell culture medium and mix by vortexing for 1&#160;s.</li>
		<li>Add 2 &#181;g of plasmid DNA (pDHsp70-Ac-P35/V5-His or pDHsp70-Ly-IAP3/V5-His or pDHsp70-Ly-IAP3-BIR/V5-His or pDHsp70-Ly-IAP3-RING/V5-His) into 100 &#181;L of serum-free cell culture medium and mix by vortexing for 1 s (Figure 2).</li>
		<li>Combine the diluted plasmid DNA and diluted cell transfection reagent (210 &#181;L), and mix by vortexing for 1 s. Incubate for 30 min at room temperature.</li>
		<li>Add 210 &#181;L of DNA transfection reagent mixture dropwise onto the cells by a P1000 pipette. Incubate at 28 &#176;C for 5 h.</li>
		<li>Replace the plating medium with 0.5 mL of cell culture medium using a P1000 pipette. Seal the 24-well plate with tape and incubate the cells at 28 &#176;C for 16 h.</li>
	</ol>
	</li>
	<li>Heat shock the transfected cells: Put the plate in a 42 &#176;C water bath (floating on the water surface). Heat for 30 min and return the 24-well plate to the 28 &#176;C incubator.</li>
	<li>Detection of protein expression
	<ol>
		<li>After 1 h or 5 h heat shock,wash the cells with 0.5 mL of 1x&#160;PBS buffer briefly three times. 
		NOTE: Dilute 10x&#160;PBS buffer to 1x PBS buffer by adding 1 mL of 10x&#160;PBS buffer to 9 mL sterilized ddH2O</li>
		<li>Lyse the cells with 40 &#181;L of 1x&#160;SDS Loading Dye by pipetting up and down. 
		NOTE: Dilute 4x&#160;SDS Loading Dye by mixing 30 &#181;L of 1x&#160;PBS buffer and 10 &#181;L of 4x&#160;SDS Sample Buffer.</li>
		<li>Heat the protein samples at 98 &#176;C for 10 min in a heat block, spin down for 1 min, and put on ice for Western blot assay.</li>
		<li>Western blot assay 
		Follow the procedure of Western blot assay from Eslami and Lujan13. Run SDS-PAGE gels14: one gel subjected to Coomassie blue staining (loading control for checking that the quantity of protein samples loaded in each well is equal in amount) and the other subjected to Western blot assay, according to Eslami and Lujan13.</li>
		<li>Detect V5-tagged fusion proteins with rabbit anti-V5 antibody (5 mg/mL) (1:5000 dilution in TBST buffer to working concentration 1 &#181;g/mL) and goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (0.8 mg/mL) (1:10000 dilution in TBST buffer to working concentration 0.08 &#181;g/mL). 
		NOTE: Adjust the percentage of polyacrylamide to 17.5% when the protein molecular weight to be &#60;17 kDa.</li>
	</ol>
	</li>
</ol>
3. Anti-apoptotic activity assay
<ol>
	<li>Gene-induced cell apoptosis: Repeat the aforementioned procedure from 2.1 to 2.3. Co-transfect 1 &#181;g of pDHsp/D-rpr/FLAG-His plasmid DNA (containing apoptosis inducer gene) with 1 &#181;g of plasmid DNA [pDHsp70/V5-His vector (negative control), pDHsp70-Ac-P35/V5-His (positive control), pDHsp70-Ly-IAP3/V5-His, pDHsp70-Ly-IAP3-BIR/V5-His or pDHsp70-Ly-IAP3-RING/V5-His, respectively.]. At 5 h post-heat shock treatment, conduct the cell viability assay (Figure 2).</li>
	<li>Chemical-induced cell apoptosis: Repeat the aforementioned procedure from 2.1 to 2.3. In step 2.1.2, plate 1 x 106 sf9 cells into each well in a 6-well plate. Transfect 4 &#181;g of plasmid DNA [pDHsp70/V5-His vector (negative control), pDHsp70-Ac-P35/V5-His (positive control), pDHsp70-Ly-IAP3/V5-His, pDHsp70-Ly-IAP3-BIR/V5-His or pDHsp70-Ly-IAP3-RING/V5-His, respectively.]. At 5 h post-heat shock, treat sf9 cells with 2mL of ActD cell culture medium for 16 h and conduct the cell viability assay (Figure 2). 
	NOTE: Minimum volume to cover one&#160; well of 6-well plate is 1 mL.</li>
	<li>Perform the above anti-apoptotic activity assay experiments, including 3.1 and 3.2 in triplicates.</li>
</ol>
4. Cell viability assay
<ol>
	<li>Wash the treated cells by adding 1 mL of 1x&#160;PBS buffer for 1 min 3 times. Resuspend the cells by pipetting 1 mL 1x&#160;PBS buffer containing 0.04% trypan blue and stain for 3 min at room temperature. Transfer the cell suspension into a 1.5 mL microtube. 
	NOTE: Dilute 0.4% trypan blue solution by mixing 9 mL of 1x&#160;PBS buffer and 1 mL 0.4% trypan blue solution.</li>
	<li>Transfer 10 &#181;L trypan blue-stained cell suspension to the hemocytometer with a P10 pipette and count the viable intact cells under a light microscope.</li>
	<li>Statistical analysis
	<ol>
		<li>Calculate the recorded data and present as the means &#177; S.D. for all counts.</li>
		<li>Analyze the data using the Student&#39;s two-tailed t-test by with Microsoft Excel. Define statistically significant data as P-value &#60;0.05.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviruses+as+gene+expression+vectors.">Baculoviruses as gene expression vectors.</a> Annual Reviews in Microbiology. 42 (1), 177-199 (1988).</li><li>Theilmann, D. A., Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., Ball, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Family+Baculoviridae.">Family Baculoviridae.</a> Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. , 1129-1185 (2005).</li><li>Nai, Y. S., Huang, Y. F., Chen, T. H., Chiu, K. P., Wang, C. H., Shields, V. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+nucleopolyhedrovirus'+taxonomic+position.">Determination of nucleopolyhedrovirus' taxonomic position.</a> Biological Control of Pest and Vector Insects. , 169-200 (2017).</li><li>Friesen, P. D., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+baculovirus+early+gene+expression.">Regulation of baculovirus early gene expression.</a> The Baculoviruses. , 141-170 (1997).</li><li>Smith, G. E., Summers, M., Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+beta+interferon+in+insect+cells+infected+with+a+baculovirus+expression+vector.">Production of human beta interferon in insect cells infected with a baculovirus expression vector.</a> Mol. Cell. Biol. 3 (12), 2156-2165 (1983).</li><li>Luckow, V. A., Summers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+the+development+of+baculovirus+expression+vectors.">Trends in the development of baculovirus expression vectors.</a> Nature Biotechnol. 6 (1), 47-55 (1988).</li><li>Jarvis, D. L., Finn, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifying+the+insect+cell+N-glycosylation+pathway+with+immediate+early+baculovirus+expression+vectors.">Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.</a> Nature Biotechnol. 14 (1996), 1288-1292 (1996).</li><li>Clem, R. J., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+programmed+cell+death+by+the+baculovirus+genes+p35+and+iap.">Control of programmed cell death by the baculovirus genes p35 and iap.</a> Mol. Cell. Biol. 14, 5212-5222 (1994).</li><li>Leu, J. H., Kuo, Y. C., Kou, G. H., Lo, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+characterization+of+an+inhibitor+of+apoptosis+protein+(IAP)+from+the+tiger+shrimp,+Penaeus+monodon.">Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon.</a> Dev. Comp. Immunol. 32, 121-133 (2008).</li><li>Zhao, Y. G., Eggleston, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+promoters+for+transient+gene+expression+in+cultured+mosquito+cells.">Comparative analysis of promoters for transient gene expression in cultured mosquito cells.</a> Insect Mol. Biol. 8 (1), 31-38 (1999).</li><li>Nai, Y. S., Yang, Y. T., Kim, J. S., Wu, C. Y., Chen, Y. W., Wang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviral+IAP2+and+IAP3+encoded+by+Lymantria+xylina+multiple+nucleopolyhedrovirus+(LyxyMNPV)+suppress+insect+cell+apoptosis+in+a+transient+expression+assay.">Baculoviral IAP2 and IAP3 encoded by Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) suppress insect cell apoptosis in a transient expression assay.</a> Appl. Entomol. Zool. 51, 305-316 (2016).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting:+Sample+Preparation+to+Detection.">Western Blotting: Sample Preparation to Detection.</a> J. Vis. Exp. (44), e2359 (2010).</li><li>. Basic Methods in Cellular and Molecular Biology. Separating Protein with SDS-PAGE Available from: <a target="_blank" href="https://www.jove.com/science-education/5058/separating-protein-with-sds-page">https://www.jove.com/science-education/5058/separating-protein-with-sds-page</a> (2017)</li><li>Vucic, D., Kaiser, W. J., Harvey, A. J., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+reaper-induced+apoptosis+by+interaction+with+inhibitor+of+apoptosis+proteins+(IAPs).">Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs).</a> Proc. Natl. Acad. Sci. USA. 94, 10183-10188 (1997).</li></ol>

The authors declare that they have no competing financial interests.

The concept of heat shock-based pDHsp/V5-His/sf9 cell system was first described by Clem et al. in 19948. Comparison of the baculoviral gene promoter (IE1) and Drosophila hsp70 showed that hsp70 had a higher efficiency in mosquito cells10. Furthermore, due to the heat shock induction, the timing of protein expression could be controlled precisely after heat shock treatment. This system was then applied for protein functional assays of shr...

Two protein expression systems have been commonly used for producing proteins: prokaryote protein expression systems (Escherichia coli gene expression system) and eukaryote protein expression systems. One popular eukaryote protein expression system is the baculovirus expression vector system (BEVS)1. Baculoviruses were first used as worldwide biological control agents of agricultural and forest pests. In the last few decades, baculoviruses were developed as biotechnological tools for protein expression vectors as well. The genomes of baculoviruses consist of double-stranded circular DNA and enveloped nucleocapsids2. To date, more than seventy-eight baculovirus isolates have been sequenced3. Based on the temporal cascade of baculoviral gene expressions in host insect cells, the gene transcription could be classified into four temporal cascades, including immediate-early, delayed-early, late, and very late genes4.
BEVSs were designated so that the very late gene promoters (i.e., polyhedron or p10 promoter) were used to drive the target genes, while the recombinant baculovirus was generated by homologous recombination. Expression of foreign proteins in insect cells by recombinant baculovirus is similar to that of mammalian proteins in post-translational modifications (suited for glycoprotein production). Thus, the baculovirus has been widely used5,6,7. However, one limitation is the presence of different N-glycosylation pathways in insect cells7.
Therefore, a new baculovirus expression system, the transient gene expression system, was developed. This system expresses foreign genes under the drive of baculoviral immediate-early promoters (ie-1 promoter) in insect cells. By using this system, the target protein can be immediately expressed under the control of ie-1 promoter while modifying the N-glycosylation pathway in insect cells, resulting in better N-linked oligosaccharides7. Moreover, baculoviral immediate-early genes are transcribed by the host cell RNA polymerase II and do not require any viral factor for activation4. Therefore, foreign proteins can be expressed in insect cells within a short time. To date, the transient gene expression system is one of the most important technologies for performing protein functional assays in the baculovirus in vitro cell culture system. The system can be applied to analyze the function of either baculovirus or foreign proteins. One of the commercially available transient gene expression systems is based on the immediate-early gene (IE) promoters of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) (OpIE2 and OpIE1 promoters).
However, the lower expression level of foreign genes in insect cells was still a problem when the OpIE promoter-based transient gene expression system was used8,9,10. Thus, another transient gene expression system was constructed based on the promoter of the Drosophila heat shock protein 70 (hsp70) gene8,9. The promoter of hsp70 works more efficiently than baculoviral IE promoter when induced by heat shock in insect cells10. In this system, the target genes were expressed under the drive of Drosophila heat shock 70 (Dhsp70) promoter. Foreign genes can be easily cloned into the transient gene expression plasmid by PCR-based cloning methods. Furthermore, the control of timing for gene expression can be performed by heat shock induction.
In this report, we follow the approach and express three different truncations of the baculoviral gene (inhibitor of apoptosis 3, iap3 from Lymantria xylina MNPV) by using the heat shock-based transient protein expression system and further apply these expressed proteins on anti-apoptotic activity analysis. This system can either express foreign proteins quickly or be further applied to the evaluation of protein anti-apoptotic activity in sf9 cells, while also having the potential to be applied to other protein activity assays.

<table><tbody><tr><td>Antibiotic-Antimycotic, 100X</td><td>Gibco</td><td>15240-062</td><td>for insect cell culture</td></tr><tr><td>Certified Foetal Bovine Serum</td><td>Bioind</td><td>04-001-1A</td><td></td></tr><tr><td>Sf-900 II SFM</td><td>Thermo Fisher</td><td>10902096</td><td>serum-free cell culture medium</td></tr><tr><td>Sf9 cells</td><td>ATCC</td><td>CRL-1711</td><td></td></tr><tr><td>25cm2 cell culture flask</td><td>Nunc, Thermo Fisher</td><td>156340</td><td></td></tr><tr><td>Inverted light microscopy</td><td>WHITED</td><td>WHITED WI-400</td><td></td></tr><tr><td>RBC HIT Competent Cell</td><td>Bioman</td><td>RH618-J80</td><td>&lt;em&gt;Escherichia coli&lt;/em&gt; (DH5&amp;alpha;)</td></tr><tr><td>L.B. Broth (Miller)</td><td>Bioman</td><td>LBL407</td><td></td></tr><tr><td>Agar, Bacteriological Grade</td><td>Bioman</td><td>AGR001</td><td></td></tr><tr><td>Zeocin</td><td>Invitrogen</td><td>ant-zn-1</td><td>selection antibiotic</td></tr><tr><td>PCR Master Mix (2X)</td><td>ThermoFisher</td><td>K0171</td><td></td></tr><tr><td>Geneaid Midi Plasmid Kit (Endotoxin Free)</td><td>Geneaid</td><td>PIE25</td><td></td></tr><tr><td>Actinomycin D</td><td>SIGMA</td><td>A9415</td><td></td></tr><tr><td>Corning 50 mL centrifuge tubes</td><td>SIGMA</td><td>CLS430829-500EA</td><td>50 mL tubes</td></tr><tr><td>Hemocytometer</td><td>Gizmo Supply Co</td><td>B-CNT-SLDE-V2</td><td></td></tr><tr><td>24-Well Multidish</td><td>Nunc, Thermo Fisher</td><td>142475</td><td>24-well plate</td></tr><tr><td>Cellfectin II Reagent</td><td>Thermo Fisher</td><td>10362100</td><td>cell transfectin reagent</td></tr><tr><td>PBS-Phosphate-Buffered Saline (10X) pH 7.4</td><td>Thermo Fisher</td><td>AM9624</td><td></td></tr><tr><td>4&amp;times;SDS Loading Dye</td><td>Bioman</td><td>P1001</td><td></td></tr><tr><td>Immobilon-P (PVDF Blotting Membranes)</td><td>Merck Milipore</td><td>IPVH00010</td><td>PVDF membranes</td></tr><tr><td>Mini Trans-Blot Cell system</td><td>BIO-RED</td><td>1703930</td><td>Blotting device</td></tr><tr><td>Ponceau S solution</td><td>SIGMA</td><td>6226-79-5</td><td></td></tr><tr><td>Anti-V5</td><td>SIGMA</td><td>V8137</td><td>rabbit anti-V5 antibody</td></tr><tr><td>Goat anti-rabbit IgG-horseradish peroxidase (HRP)</td><td>Jackson</td><td>111-035-003</td><td></td></tr><tr><td>Tween 20</td><td>Merck</td><td>817072</td><td></td></tr><tr><td>6-Well Multidish</td><td>Nunc, Thermo Fisher</td><td>145380</td><td></td></tr><tr><td>0.4 % trypan blue solution</td><td>AMRESCO</td><td>K940-100ML</td><td></td></tr><tr><td>P10 pipetman</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>P1000 pipetman</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Tape</td><td>Symbio</td><td>PPS7</td><td>24 well tape ( 19 mm&amp;times;36 M)</td></tr></tbody></table>

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.

The full length and other two truncations (BIR and RING domains) of Ly-IAP3 from LyxyMNPV were overexpressed in sf9 cells, based on the heat shock-based pDHsp/V5-His/Spodoptera frugiperda cell (sf9 cell) system. The pDHsp/V5-His contained a Drosophila heat shock protein promoter, which drives downstream gene expression at a temperature of 42 &#176;C condition by using cellular transcriptional factors and the translation system (Figure 1)<sup...

Watch this Scientific Journal Video about Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay at JoVE.com

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.

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 ...

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12.2K Views.  National Ilan University. 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.

Video: Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

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 ...

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Biochemistry

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Bioengineering

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. ...

Biology

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 ...

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.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

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.

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 of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

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Chapters in this video

Controllable Ion Channel Expression through Inducible Transient Transfection

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

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

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

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

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