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Cloning, expression and purification of small subunit of Exodeoxyribonuclease VII from Vancomycin Resistant Staphylococcus aureus (VRSA).
EP34637
Poster Title: Cloning, expression and purification of small subunit of Exodeoxyribonuclease VII from Vancomycin Resistant Staphylococcus aureus (VRSA).
Submitted on 01 Jan 2021
Author(s): Faryal Ashraf, Atia-tul-Wahab, M. Iqbal Choudhary
Affiliations: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi
This poster was presented at 6th International Symposium-cum-Training Course on Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi
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Poster Information
Abstract: Exodeoxyribonuclease VII (ExoVII) is an enzyme that catalyzes the bidirectional exonucleolytic cleavage of single stranded DNA. ExoVII comprises of two subunits i.e. large XseA and small XseB [1]. It is one of the key players of DNA repair and recombination processes occur in bacteria. Despite having central role in DNA repair and recombination, the target was remained ignore since its first published data in 1974 by Chase and Richardson on ExoVII of E.coli [2,3]. ExoVII has significant potential to serve as drug target.
The present work is focused on the cloning, expression and purification strategies of small subunit XseB of ExoVII from Vancomycin Resistant Staphylococcus aureus (VRSA) strain Mu50. The gene xseB encodes 231 bp gene products which was cloned in pSpeedET vector and transformed to E.coli DH5α strain. This was followed by expression of construct in E.coli BL21DE3 cells. The expressed product of 8.7 kDa was purified from
rest of the bacterial proteins using Ni-NTA resin through affinity chromatography. Then further oligomeric nature of protein was determined using size exclusion chromatography (75pg superdex). However, 1 H, 15 N HSQC spectra indicated the folding of protein.
Summary: This work describes the purification studies of small subunit of Exodeoxyribonuclease VII (ExoVII) using recombinant DNA technology.References: 1. Poleszak, K., Kaminska, K. H., Dunin-Horkawicz, S., Lupas, A., Skowronek, K. J.,
& Bujnicki, J. M. (2012). Delineation of structural domains and identification of
functionally important residues in DNA repair enzyme exonuclease VII. Nucleic
acids research, 40(16), 8163-8174.
2. Chase, J. W., & Richardson, C. C. (1974). Exonuclease VII of Escherichia coli
PURIFICATION AND PROPERTIES. Journal of Biological Chemistry, 249(14),
4545-4552.
3. Chase, J. W., & Richardson, C. C. (1974). Exonuclease VII of Escherichia coli
MECHANISM OF ACTION. Journal of Biological Chemistry, 249(14), 4553-
4561.
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