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.
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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
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Acknowledgements
The authors would like to extend their gratitude to Department of Life Sciences, University of Calicut, India, for providing necessary facilities.
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This work was supported by Innovative Research Project, University of Calicut.
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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.
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Sereena, M.C., Sebastian, D. Cloning, Expression and Characterization of the Anticancer Protein Azurin from an Indigenous Strain Pseudomonas aeruginosa SSj. Int J Pept Res Ther 26, 1223–1230 (2020). https://doi.org/10.1007/s10989-019-09924-1
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DOI: https://doi.org/10.1007/s10989-019-09924-1