Cloning, expression and purification of Pseudomonas aeruginosa azurin, a small redox protein and its application in molecular electronics

INTRODUCTION Redox metalloproteins are getting considerable interest nowadays due to their applications in creating hybrid sub micrometre-sized electronic components for biosensors, transistors and memory devices. These... [ view full abstract ]

INTRODUCTION

Redox metalloproteins are getting considerable interest nowadays due to their applications in creating hybrid sub micrometre-sized electronic components for biosensors, transistors and memory devices. These bio-nanodevices overcome the limitations of silicon-based devices like tunnelling effects, efficiency, and heat. These are operationally simple, cost efficient and suitable for real time detection. Azurin from Pseudomonas aeruginosa is an oxidoreductase, which possess an inherent, efficient electron transfer capability, occurring at single electron level in a very fast and directional way. Also, this has been proved to stabilize p53 tumor suppressor protein and hence, useful in cancer therapy. Azurin has a great potential to be used in the molecular electronics. But the purification of this protein is difficult and require multiple steps which result in low yield. We have cloned the protein and purify it in single step and got better yield of the protein. We have confirmed its electron transfer properties with cyclic voltammetry and conductive AFM.

METHOD
Azurin gene was cloned and expressed in Escherichia coli and purified by Ni-NTA (Nickel-Nitrilo acetic acid) chromatography. Recombinant azurin was characterized using UV-Visible, FTIR, Circular Dichroism and Raman Spectroscopy. Azurin was immobilized directly on gold via thiol linkage and on silicon and mica using 3-mercaptopropyl trimethoxysilane as linker. Conductive atomic force microscopy was also done to study the morphology and conductivity of azurin layer on different substrates.

RESULTS AND DISCUSSION

Azurin gene was successfully cloned and expressed in good amount. Pure band of azurin was obtained after single step of Ni-NTA purification. The excellent current density of azurin layer on gold was observed in cyclic voltammetry experiments. The sharp oxidation and reduction potentials peaks were as reported for the wild type azurin. In conductive atomic force microscopy the current observed was higher in the azurin immobilized sample than the blank one.

CONCLUSION
We were successful in getting the pure active protein in very good quantity which can be used directly for electronic studies. Same protein can be used as the acceptor and donor of electrons. Its small size and efficient electron transfer capability make it a desirable molecule for the development of biosensors.