Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

• Published in: PloS one 2015-09, Vol.10 (9), p.e0139248
• Main Authors: Rothan, Hussin A, Ambikabothy, Jamunaa, Abdulrahman, Ammar Y, Bahrani, Hirbod, Golpich, Mojtaba, Amini, Elham, A Rahman, Noorsaadah, Teoh, Teow Chong, Mohamed, Zulqarnain, Yusof, Rohana
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anticancer properties; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacology; Biocompatibility; Biological activity; Buffers; C-Terminus; Cancer; Cancer therapies; Cancer treatment; Cationic peptides; Cell Line; Cell Proliferation - drug effects; Cell Survival - drug effects; Chemotherapy; Dengue fever; Design; Dose-Response Relationship, Drug; Doxorubicin; Doxorubicin - pharmacology; Drug delivery systems; Drug Synergism; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fusion protein; Glutathione; Hep G2 Cells; Humans; Immunoblotting; Inclusion bodies; Inclusion Bodies - metabolism; Kinases; Mass production; MCF-7 Cells; Medical research; Medicine; Methods; Microscopy, Confocal; Molecular Sequence Data; Oxides; Peptides; Peptides - metabolism; Peptides - pharmacology; Physiological aspects; Physiology; Protein folding; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Recombinant proteins; Ribosome Inactivating Proteins, Type 2 - genetics; Ribosome Inactivating Proteins, Type 2 - metabolism; Ribosome Inactivating Proteins, Type 2 - pharmacology; Science; Solubilization; Toxicity; Tumors; Urea
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0139248

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.