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

Summary

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.

Abstract

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.

Introduction

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

Protocol

1. Preparations

1. Insect cell culture
   1. Prepare 50 mL of cell culture medium. To do so, add 500 µL of antibiotics (Amphotericin B = 0.25 µg/mL, Penicillin = 100 unit/mL, Streptomycin = 100 µg/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 °C for 30 min in a water bath before use.
   2. 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. T....

Results

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 °C condition by using cellular transcriptional factors and the translation system (Figure 1)

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibiotic-Antimycotic, 100X	Gibco	15240-062	for insect cell culture
Certified Foetal Bovine Serum	Bioind	04-001-1A
Sf-900 II SFM	Thermo Fisher	10902096	serum-free cell culture medium
Sf9 cells	ATCC	CRL-1711
25cm2 cell culture flask	Nunc, Thermo Fisher	156340
Inverted light microscopy	WHITED	WHITED WI-400
RBC HIT Competent Cell	Bioman	RH618-J80	Escherichia coli (DH5α)
L.B. Broth (Miller)	Bioman	LBL407
Agar, Bacteriological Grade	Bioman	AGR001
Zeocin	Invitrogen	ant-zn-1	selection antibiotic
PCR Master Mix (2X)	ThermoFisher	K0171
Geneaid Midi Plasmid Kit (Endotoxin Free)	Geneaid	PIE25
Actinomycin D	SIGMA	A9415
Corning 50 mL centrifuge tubes	SIGMA	CLS430829-500EA	50 mL tubes
Hemocytometer	Gizmo Supply Co	B-CNT-SLDE-V2
24-Well Multidish	Nunc, Thermo Fisher	142475	24-well plate
Cellfectin II Reagent	Thermo Fisher	10362100	cell transfectin reagent
PBS-Phosphate-Buffered Saline (10X) pH 7.4	Thermo Fisher	AM9624
4×SDS Loading Dye	Bioman	P1001
Immobilon-P (PVDF Blotting Membranes)	Merck Milipore	IPVH00010	PVDF membranes
Mini Trans-Blot Cell system	BIO-RED	1703930	Blotting device
Ponceau S solution	SIGMA	6226-79-5
Anti-V5	SIGMA	V8137	rabbit anti-V5 antibody
Goat anti-rabbit IgG-horseradish peroxidase (HRP)	Jackson	111-035-003
Tween 20	Merck	817072
6-Well Multidish	Nunc, Thermo Fisher	145380
0.4 % trypan blue solution	AMRESCO	K940-100ML
P10 pipetman	Gilson	F144802
P1000 pipetman	Gilson	F123602
Tape	Symbio	PPS7	24 well tape ( 19 mm×36 M)