These authors contributed equally to this work.
Gene cloning, bacterial expression, and purification of calreticulin from Anopheles stephensi (AsCRT)
Article first published online: 17 JAN 2012
© 2012 The Authors. Entomological Research © 2012 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd
Volume 42, Issue 1, pages 28–34, January 2012
How to Cite
BORHANI, N., HEIDARI, M. and BASSERI, H. R. (2012), Gene cloning, bacterial expression, and purification of calreticulin from Anopheles stephensi (AsCRT). Entomological Research, 42: 28–34. doi: 10.1111/j.1748-5967.2011.00357.x
- Issue published online: 17 JAN 2012
- Article first published online: 17 JAN 2012
- Received 6 June 2011; accepted 18 October 2011.
- Anopheles vectors;
- recombinant GST-calreticulin;
- Western blotting
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.