Name: transient expression of foreign genes in insect cells (sf9) for protein functional assay
Uploaded: 2018-02-22T13:00:00
Description: Watch this Scientific Journal Video about Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay at JoVE.com

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: Noctui...

<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 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>Escherichia coli (DH5&#945;)</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&#215;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&#215;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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