Design and development of an novel anticancer peptide from human gut microbiome by using recombinant protein engineering

Human microbiota is a microbial community that lives on and in the human body. It has received considerable attention and research ef-forts over the past decade because it exerts a major impact on human health, from metabolism to immunity. In order to leverage the close associations between microbes and their host, many studies have identified and screened therapeutic drugs from the human microbiome with a focus on the gut microbiome. 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). The results from these steps show that the plasmid pDT008 allows Cnazu8 to express in fusion with GST-6xHis-TEV in E. coli. In particularly, the optimal conditions for expression of the fusion peptide GST-6xHis-TEV-Cnazu8 with the expected molecular weight of 36.7 kDa are 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 disrup-tion is developed to increase the solubility of the desired proteins. Finally, protein purification is performed in a HisPur Ni-NTA column (Qiagen) which results in a relatively low amount of desired fusion proteins. Thus, the optimization of protein purification and anticancer bioassays of this peptide are required for further studies to develop Cnazu8 as a novel therapeutic drug candidate against cancer.