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Cloning, Expression and Characterization of the Anticancer Protein Azurin from an Indigenous Strain Pseudomonas aeruginosa SSj

  • M. C. Sereena
  • Denoj SebastianEmail author
Article
  • 18 Downloads

Abstract

Azurin is a novel anticancer protein, mainly produced from Pseudomonas aeruginosa and few other Gram-negative bacteria. In this study, azurin gene (azu) from a native Pseudomonas aeruginosa strain SSj was amplified using PCR with specific primers. The azurin gene (418 bp) was sequenced and submitted in Genbank. The PCR amplicon was digested with BamHI and HindIII and inserted into expression vector pET-22b(+). The recombinant protein was expressed in Escherichia coli BL21 (DE3) after induction with IPTG. Protein purification was done using Ni NTA agarose. The SDS-PAGE analysis of purified samples with Coomassie brilliant blue showed a band with an apparent molecular weight of about 14 kDa. Structural characterization of purified protein was done by FTIR, MALDI-TOF and LC–MS/MS analysis.

Keywords

Azurin BL21 (DE3) pET-22b(+) MALDI-TOF LC–MS/MS Anticancer protein 

Abbreviations

PCR

Polymerase chain reaction

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

IPTG

Isopropyl β-d-1-thiogalactopyranoside

Ni NTA

Nickel-nitrilotriacetic acid

FTIR

Fourier-transform infrared spectroscopy

MALDI-TOF

Matrix assisted laser desorption/ionization time of flight

LC–MS/MS

Liquid chromatography with tandem mass spectrometry

Notes

Acknowledgements

The authors would like to extend their gratitude to Department of Life Sciences, University of Calicut, India, for providing necessary facilities.

Author Contributions

This work was carried out in collaboration between both authors. Author DS designed the study and done supervision and manuscript correction. Author MCS managed the literature searches, manuscript work and the experimental process. Both authors read and approved the final manuscript.

Funding

This work was supported by Innovative Research Project, University of Calicut.

Compliance with Ethical Standards

Conflict of interest

Authors have no conflict of interest regarding the publication of paper.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Life SciencesUniversity of CalicutThenjipalamIndia

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