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Biochemistry

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

Published: August 23rd, 2018

DOI:

10.3791/58106

1Department of Entomology and Plant Pathology, Auburn University

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 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 MdαE7 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 (MdαE7-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 MdαE7 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 MdαE7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Insecticide resistance is currently a major issue of house fly control worldwide1,2. Efforts to determine the mechanism of insecticide resistance facilitates better understanding of this issue and thus provide novel strategies to effectively prevent or minimize the spread of resistance development3. Carboxylesterases, as one of the major detoxification enzymes, have attracted a lot of attention for their roles in sequestering and metabolizing insecticides in various insects4,5,6. Our previous s....

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1. Expression and Isolation of Target Proteins with a Baculovirus-mediated Insect Cell Expression System

  1. Directionally clone blunt-ended PCR products of target proteins from house flies.
    1. Design PCR primers of green fluorescent protein (GFP) and the house fly MdαE7 gene based on their sequences and the special requirements of the chosen vector (Table 1).
    2. Use a thermostable, proofreading DNA polymerase and primers from step 1.1.1 to perform a 150 µL PCR rea.......

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The cell viability toward different permethrin treatments (MTT assay)

The cytotoxicity of permethrin was examined in MdαE7-recombinant baculovirus infected Sf9 cells (experimental group) and CAT-recombinant baculovirus (provided by baculovirus infected kit) infected cells (control groups). The enhanced cell tolerances to permethrin in MdαE7 expressing cells strongly support the metabolic roles of this carboxylesterase aga.......

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In recent decades, heterologous expression systems have been widely used to express and isolate large amounts of proteins, thus allowing biochemical and functional determination and characterization of enzymes in vitro. To date, several different model systems including Escherichia coli, Pichia pastoris, Sacccharomyces cerevisiae, and Spodoptera frugiperda have been adapted for recombinant protein expression, and the choice of the in vitro system is crucial for large .......

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Name Company Catalog Number Comments
Q5 High-Fidelity DNA Polymerase New England Biolabs inc. M0491L
QIAquick Gel Extraction Kit QIAGEN 28704
pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli Invitrogen by life technology K240020 S.O.C medium and universal M13 sequence primers were included in this kit.
PureLink HiPure Plasmid Miniprep Kit Invitrogen by life technology K210002
Gateway LR Clonase II Enzyme mix for BaculoDirectTM Kits Invitrogen by life technology 11791-023
BaculoDirect C-Term Linear DNA Transfection Kit Invitrogen by life technology 12562-019 Cellfectin transfection reagent and ganciclovir were included in this kit
pENTR-CAT plasmid Invitrogen by life technology Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL
Heat inactivated Fetal Bovine Serum, Certified Gibco by Life Technologies 10082-139
Sf9 cells in Sf-900 III SFM Gibco by Life Technologies 12659017
Insect Cell-PE LB Insect Cell Protein Extraction & Lysis Buffer G Biosciences by A Geno Technology Inc 786-411
Sf-900 III SFM (1×) Serum Free Medium Complete Gibco by Life Technologies 12658-019
Grace's Insect Medium, unsupplemented Gibco by Life Technologies 11595030
Permethrin (isomers) analytical standard SUPELCO by Solutions WithinTM 442748
Methanol (analytical graded) Sigma-Aldrich 67-56-1
Acetonitrile (analytical graded) Sigma-Aldrich 75-05-8
GHP Acrodisc 25 mm Syringe Filters with 0.45 μm GHP Membrane (HPLC Certified) Pall Life Sciences 21890388
Alliance Waters 2695 HPLC System Waters
T100 Thermal Cycle Bio-Rad Laboratories Inc. 1861096
Nanodrop 2000/2000c Spectrophotometers ThermoFisher Scientific ND2000CLAPTOP
Cytation 5 Cell Imaging Multi-Mode Reader BioTek

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