Expression, purification and functional analysis of an odorant binding protein AaegOBP22 from Aedes aegypti.

Mosquitoes that act as disease vectors rely upon olfactory cues for host-seeking, mating, blood feeding and oviposition. To reduce the risk of infection in humans, one of the approaches focuses on mosquitoes' semiochemical system in the effort to disrupt undesirable host-insect interaction. Odorant binding proteins (OBPs) play a key role in mosquitoes' semiochemical system. Here, we report the successful expression, purification of an odorant binding protein AaegOBP22 from Aedes aegypti in heterologous system. Protein purification methods were set up by Strep-Tactin affinity binding and size-exclusion chromatography. Analysis by SDS-PAGE and mass spectrum revealed the protein's purity and molecular weight. Circular dichroism spectra showed the AaegOBP22 secondary structure had a pH dependent conformational change. The protein functions of AaegOBP22 were tested by fluorescent probe 1-NPN binding assays and ligands competitive binding assays. The results show AaegOBP22 proteins have characteristics of selective binding with various ligands.

[1]  V. Loeschcke,et al.  Dopamine levels in the mosquito Aedes aegypti during adult development, following blood feeding and in response to heat stress. , 2006, Journal of insect physiology.

[2]  H. Biessmann,et al.  The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes , 2010, PloS one.

[3]  Long Zhang,et al.  Binding specificity of locust odorant binding protein and its key binding site for initial recognition of alcohols. , 2009, Insect biochemistry and molecular biology.

[4]  C. Smartt,et al.  Expression of a Novel Member of the Odorant-Binding Protein Gene Family in Culex nigripalpus (Diptera: Culicidae) , 2009, Journal of medical entomology.

[5]  Zainulabeuddin Syed,et al.  Mosquitoes smell and avoid the insect repellent DEET , 2008, Proceedings of the National Academy of Sciences.

[6]  L. Field,et al.  Identification of odorant‐binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses , 2008, Insect molecular biology.

[7]  S. Wikel,et al.  Differential expression of Aedes aegypti salivary transcriptome upon blood feeding , 2009, Parasites & Vectors.

[8]  Arne Skerra,et al.  The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins , 2007, Nature Protocols.

[9]  H. Biessmann,et al.  Specific interactions among odorant‐binding proteins of the African malaria vector Anopheles gambiae , 2006, Insect molecular biology.

[10]  W. Xu,et al.  Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive “Lid” , 2009, PloS one.

[11]  S. Turillazzi,et al.  Exploring Proteins in Anopheles gambiae Male and Female Antennae through MALDI Mass Spectrometry Profiling , 2008, PloS one.

[12]  P. Pelosi,et al.  Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts , 2005, Cellular and Molecular Life Sciences CMLS.

[13]  J. Choi,et al.  Secretory and extracellular production of recombinant proteins using Escherichia coli , 2004, Applied Microbiology and Biotechnology.

[14]  W. Xu,et al.  Reverse and Conventional Chemical Ecology Approaches for the Development of Oviposition Attractants for Culex Mosquitoes , 2008, PloS one.

[15]  P. Pelosi,et al.  Multiple functions of an odorant-binding protein in the mosquito Aedes aegypti. , 2008, Biochemical and biophysical research communications.

[16]  W. Leal,et al.  Genome Analysis and Expression Patterns of Odorant-Binding Proteins from the Southern House Mosquito Culex pipiens quinquefasciatus , 2009, PloS one.

[17]  Evgeny M. Zdobnov,et al.  Genome Sequence of Aedes aegypti, a Major Arbovirus Vector , 2007, Science.

[18]  R. Vogt,et al.  Antennal expressed genes of the yellow fever mosquito (Aedes aegypti L.); characterization of odorant-binding protein 10 and takeout. , 2005, Insect biochemistry and molecular biology.

[19]  Jing-Jiang Zhou,et al.  "Plus-C" odorant-binding protein genes in two Drosophila species and the malaria mosquito Anopheles gambiae. , 2004, Gene.

[20]  C. Nespoulous,et al.  Odorant binding and conformational changes of a rat odorant-binding protein. , 2004, Chemical senses.

[21]  A. Cornel,et al.  Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants , 2010, Journal of Chemical Ecology.

[22]  M. Wogulis,et al.  The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism. , 2006, Biochemical and biophysical research communications.

[23]  H. Breer,et al.  Olfactory receptors: molecular basis for recognition and discrimination of odors , 2003, Analytical and bioanalytical chemistry.

[24]  W. Takken,et al.  Olfactory regulation of mosquito-host interactions. , 2004, Insect biochemistry and molecular biology.

[25]  A. Kuhn,et al.  Evidence for a loop-like insertion mechanism of pro-Omp A into the inner membrane of Escherichia coli. , 1994, European journal of biochemistry.

[26]  E. Guittet,et al.  Structural basis of the broad specificity of a general odorant-binding protein from honeybee. , 2009, Biochemistry.

[27]  J. Krzywinski,et al.  Analysis of the complete mitochondrial DNA from Anopheles funestus: an improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution. , 2006, Molecular phylogenetics and evolution.

[28]  Loïc Briand,et al.  Evidence of an odorant-binding protein in the human olfactory mucus: location, structural characterization, and odorant-binding properties. , 2002, Biochemistry.

[29]  J. Beechem,et al.  Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. , 2002, Analytical biochemistry.

[30]  Y. Lee,et al.  Roles of the signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi. , 1997, The Biochemical journal.