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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- 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
References
Ali MA, Mohamed MS, Sedek RG (2017) Can metal replacement increase the antitumor activity of azurin? (in vitro study). Gen Med Open 1(2):1–7. https://doi.org/10.15761/GMO.1000111
Canters G (1987) Cloning and sequencing of the azurin gene from Pseudomonas-aeruginosa. FEBS Lett 212(1):168–172. https://doi.org/10.1016/0014-5793(87)81579-X
Chakrabarty AM, Bernardes N, Fialho AM (2014) Bacterial proteins and peptides in cancer therapy: today and tomorrow. Bioengineered 5(4):234–242. https://doi.org/10.4161/bioe.29266
Elliott A, Ambrose EJ (1950) Structure of synthetic polypeptides. Nature 165(4206):921–922
Fukuo T, Kubota N, Kataoka K, Nakai M, Suzuki S, Arakawa R (1998) Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry analysis of blue copper proteins Azurin and mavicyanin. Rapid Commun Mass Spectrom 12(23):1967–1971
Hashimoto W, Ochiai A, Hong CS, Murata K, Chakrabarty AM (2015) Structural studies on Laz, a promiscuous anticancer Neisserial protein. Bioengineered 6(3):141–148. https://doi.org/10.1080/21655979.2015.1022303
Horio T (1958) Terminal oxidation system in bacteria. J Biochem 45(4):267–279
Karlsson BG, Pascher T, Nordling M, Arvidsson RH, Lundberg LG (1989) Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli. FEBS Lett 246(1–2):211–217
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
McCormack AL, Schieltz DM, Goode B, Yang S, Barnes G, Drubin D, Yates JR (1997) Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal Chem 69(4):767–776. https://doi.org/10.1021/ac960799q
Mohamed MS, Fattah SA, Mostafa HM (2010) Azurin as antitumor protein and its effect on the cancer cell lines. Curr Res J Biol Sci 2(6):396–401
Ramachandran S, Singh M, Mandal M (2012) Purification of azurin from Pseudomonas Aeuroginosa. Chromatography Leonardo de Azevedo Calderon, IntechOpen. https://doi.org/10.5772/51891
Sankar R, Siddik S, Abhijit M, Mahitosh M (2011) Azurin synthesis from Pseudomonas Aeruginosa MTCC 2453, properties, induction of reactive oxygen species, and p53 stimulated apoptosis in breast carcinoma cells. J Cancer Sci Ther 03:104–111. https://doi.org/10.4172/1948-5956.1000069
Surewicz WK, Szabo AG, Mantsch HH (1987) Conformational properties of azurin in solution as determined from resolution-enhanced Fourier-transform infrared spectra. Eur J Biochem 167(3):519–523
Sutherland IW, Wilkinson JF (1963) Azurin: a copper protein found in Bordetella. J Gen Microbiol 30:105–112. https://doi.org/10.1099/00221287-30-1-105
Valli S (2018) Bioinformatic study of an antitumor protein, azurin. Asian J Pharm Clin Res 11(6):169. https://doi.org/10.22159/ajpcr.2018.v11i6.23339
Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23(6):316–320
Winnepenninckx B, Backeljau T, De Wachter R (1993) Extraction of high molecular weight DNA from molluscs. Trends Genet 9(12):407
Wood PM (1978) Periplasmic location of the terminal reductase in nitrite respiration. FEBS Lett 92(2):214–218
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