Cloning and Expression of TRYP6 Gene from Leishmania major (MRHO/IR/75/ER)

Abstract

Background: Leishmania, needs to detoxify the macrophage derived potent peroxides (H2O2). Tryparedoxin path­way contains tryparedoxin peroxidase (TSA or TRYP). The aim of the study was to detect the full-length gene se­quence and its encoded protein of the LmTRYP6 gene (EU251502), and comparison the gene sequence with LmTRYP6 (LmjF15.1140), another previously reported member of this gene family. Methods: L.major (MRHO/IR/75/ER) promastigotes were cultured, DNA and RNA were extracted and the inter­ested gene was amplified using PCR and RT-PCR methods. PCR/ RT-PCR fragments were purified and cloned first in pTZ57R/T and then in pET15b expression vector. The expressed protein was verified using western blot method. Char­acterization of the expressed protein was performed bioinformatically. Results: Molecular evaluation revealed that the cloned LmTRYP6 gene (EU251502) encoded a predicted 184 amino acid long protein with a theoretical isoelectric point of 6.1101. Alignment showed a number of changes in amino acid composition including the replacement of highly conserved Trp177 by Cys in LmTRYP6 (ABX26130). Conclusion: So far no study has been done on this group, i.e. TRYP6 gene, from tryparedoxin peroxidase family. The low homology with LmTRYP6 (LmjF15.1140) and vast array of differences observed in the gene under study (LmTRYP6; EU251502) could open new windows in the field of anti-Leishmania combat. Based on its important role in the viability and successful establishment of the parasite in the host organism it looks to be very good candi­date for vaccine development and any other sort of novel drug development.

Keywords

• Tryparedoxin peroxidase
• L. major
• Peroxiredoxin
• TRYP6
• Cloning