Construction And Application Of Azurin Expression Vector Characterized With High-performance Secretion

Azurin, a 14.6kDa bacterial redox protein with chimeric copper ion, is composed of 128 amino acids and secreted from Pseudomonas aeruginosa. It plays an important role in the redox and electron transport during respiration in microorganisms. But in recent years, some reports demonstrated that azurin could inhibit the growth of several tumor cells, such as J774 tumor cell, melanoma UISO-Mel-2 cell, human breast cancer MCF-7 cell and osteosarcoma U2OS cell. Azurin exerts the anti-proliferation by inducing cell apoptosis. It can form a complex with the tumor suppressor protein p53, stabilizing it and raising its intracellular level. The accumulation of p53 in cell can lead to the cell apoptosis.In this study, the azurin gene was amplified from Pseudomonas putida KT2440 by PCR. We employed Red/ET homologous recombination technology to construct the typeⅠsecreting expression vector with E. coli a-hemolysin and the typeⅡsecreting expression vector. The pelB signal peptide gene was coexpressed with azurin in typeⅡsystem to lead the protein going through cell membrane. Both of them were transformed into E.coli (DE3) respectively and induced by tetracycline. According to the results of sequence alignment, the azurin gene from Pseudomonas putida KT2440 and Pseudomonas aeruginosa PAO1 has 81% homologous sequence. The azurin can be more efficiently secreted into culture medium in typeⅡexpression vector in comparison with typeⅠby SDS-PAGE and western Blot assays. Azurin was purified from the supernatant of E.coli typeⅡsecretion system by nickel-affinity chromatography, and the purification was proved efficiently by SDS-PAGE assay. The purified azurin was used to treat melanoma B16 cells and 4T1 cells. The results of MTT showed that azurin had a significant inhibition effect on the cancer cells. The assay of DNA Ladder demonstrated that the cancer cells were involved in apoptotic process after treatment. The typeⅡvector allows azurin gene expressing and secreting efficiently in E.coli. This is a potential approach in the application of azurin in tumor therapeutic research and the expression system construction of micromolecule proteins.