Design and Development of a Novel Anticancer Peptide from Human Gut Microbiome by Using Recombinant Protein Engineering

Abstract

Human microbiota is a microbial community that lives on and in the human body. It has received considerable attention and research efforts over the past decade because it exerts a major impact on human health, from metabolism to immunity. In a recent study, we identified novel anticancer Azurin-like peptides from the human gut microbiome using combined molecular biology and bioinformatics based approaches. Herein, we present the cloning, expression and partial purification of one of these peptides as a case study towards the design and development of novel anticancer peptide drugs by the use of recombinant protein engineering. Firstly, the vector pET42a(+) is used for the cloning of a peptide Cnazu8 encoded by p2seq12 (cnazu8) from Clostridium nexile DSM 1718 in E. coli OmniMAX. Secondly, this vector is further used for expression in E. coli BL21 (DE3). Finally, protein purification is performed in a HisPur Ni-NTA column (Thermo). The results show that the plasmid pDT008 allows Cnazu8 to express in fusion with GST-6xHis-TEV in E. coli. The optimal conditions for expression of the fusion peptide GST-6xHis-TEV-Cnazu8 (36.7 kDa) include IPTG at 0.05 mM and the temperature at 37 °C. However, most of the expected proteins are expressed in the insoluble forms. Thus, a sonication method for cell disruption is developed to increase the solubility of the desired proteins. The purification results in a relatively low amount of desired fusion proteins. Thus, the purification optimization and anticancer bioassays of Cnazu8 are required to further consider as a novel anticancer drug candidate.