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Gene cloning, bacterial expression, and purification of calreticulin from Anopheles stephensi (AsCRT)

Authors

  • Nahid BORHANI,

    1. Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
    2. Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
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    • These authors contributed equally to this work.

  • Mansour HEIDARI,

    1. Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
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    • These authors contributed equally to this work.

  • Hamid R. BASSERI

    Corresponding author
    1. Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
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Hamid Reza Basseri, Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Pour Sina Ave, Tehran, Iran. Email: basserih@tums.ac.ir

Abstract

During the invasion of Plasmodium ookinetes to the mosquito midgut epithelium, several proteins or glycoproteins are involved. Recent study has shown that the calreticulin (CRT) of the midgut from Anopheles albimanus can bind to the protein receptor Pvs25 on surface of Plasmodium vivax ookinetes. Thus, in order to get more insight into the potential roles of Anopheles stephensi calreticulin (AsCRT) in the midgut, we amplified and cloned the full-length of calreticulin coding sequence from Anopheles stephensi. The AsCRT consists of 1221 bp nucleic acids with one open reading frame (ORF) encoding 406 amino acids and an apparent molecular weight around 46 KDa. Subsequently, the recombinant calreticulin as Glutathione S-transferase (GST) fusion in pGEX −6p-1 expression vector (GST-AsCRT) was produced in the prokaryotic system under optimum conditions. GST-AsCRT fusion protein has a molecular weight around 73 KDa. The recombinant protein was detected by Western blotting using a rabbit anti-GST polyclonal antibody. Here, we report via single protein purification procedure using MagneGST beads, 25 mg of the recombinant protein was obtained per liter of bacterial culture. This is the first report describing the heterologous expression of Anopheles stephensi calreticulin in the prokaryotic system. The production of this recombinant protein will now allow us to further investigate AsCRT molecular protein analyses, characterization of physiochemical properties, as well as interaction between calreticulin and plasmodium protein surface.

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